Comparison of three fecal steroid metabolites for pregnancy detection used with single sampling in bighorn sheep (Ovis canadensis)

We conpared three fecal steroid metabolite assays for their usefulness in detecting pregnalcy among free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from Bighorn Canyon National Recreation Area, Wyoming and Montana (USA) and captive bighorn ewes at ZooMontana in Billings, Montana. Fecal samples were collected from 11 free-ranging, radio-collared bighorn ewes in late January-May 2001 and from 20 free-ranging, radio-collared ewes in late March to mid-May 2002. Free-ranging ewes were monitored the following spring to determine whether or not they lambed. In addition, two captive ewes were studied at ZooMontana. With three exceptions, free-ranging bighorn ewes that produced lambs had nonspecific progesterone metabolite (iPdG) levels of >1800 ng/g feces and iPdG levels >7000 ng/gm feces when samples were collected between early March and mid-May. Samples collected earlier in the year were inconclusive. One false negative was suspected to be the result of sample collection error. Of the captive ewes, nonspecific pregnanediol-3alpha-glucuronide (PdG) and iPdG followed a predictable curve over the course of the 180-day pregnancies. We conclude that estrone conjugates are not useful in diagnosing pregnancy; however, fecal steroid analysis of PdG and iPdG can be used to accurately determine pregnancy and reproductive function in bighorn sheep. This holds great potential as a noninvasive technique for understanding the role of reproductive disease in wild bighom sheep.     

Predator-induced natural selection on temperament in bighorn ewes

Temperament traits in animals may have important fitness consequences, but have received little attention from ecologists or evolutionary biologists. A few studies have linked variation in temperament with fitness, but none has measured selection on temperament traits. We estimated the strength of selection on female boldness and docility on bighorn sheep ewes, Ovis canadensis. The Ram Mountain population experienced little predation pressure during the first 25 years of study, then 2 years (1997 and 1998) of frequent predation by cougars, Puma concolor, during which adult ewe mortality almost tripled over the long-term average, to 27% a year. During years of high predation, we found moderate selection favouring bold ewes, and age-specific selection on docility. Old ewes appeared more vulnerable to predation than young ewes. In contrast, no evidence of selection on temperament traits was observed during 2 years of low predation (1996 and 1999). These results suggest predator-induced selection favouring bold and nondocile ewes. Leadership was highly correlated with age and may increase the risk of predator encounter. Leadership alone, however, could not explain the higher vulnerability of old ewes to predation. Cougar predation on bighorn sheep occurs sporadically and unpredictably, probably because individual cougars often are prey specialists. Cougar predation may have limited microevolutionary effects on temperament in bighorn sheep, because it mostly affects ewes near the end of their reproductive life span and because of potential countervailing selection on boldness and docility. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.      

Use of Exposure History to Identify Patterns of Immunity to Pneumonia in Bighorn Sheep (Ovis canadensis)

Individual host immune responses to infectious agents drive epidemic behavior and are therefore central to understanding and controlling infectious diseases. However, important features of individual immune responses, such as the strength and longevity of immunity, can be challenging to characterize, particularly if they cannot be replicated or controlled in captive environments. Our research on bighorn sheep pneumonia elucidates how individual bighorn sheep respond to infection with pneumonia pathogens by examining the relationship between exposure history and survival in situ. Pneumonia is a poorly understood disease that has impeded the recovery of bighorn sheep (Ovis canadensis) following their widespread extirpation in the 1900s. We analyzed the effects of pneumonia-exposure history on survival of 388 radio-collared adults and 753 ewe-lamb pairs. Results from Cox proportional hazards models suggested that surviving ewes develop protective immunity after exposure, but previous exposure in ewes does not protect their lambs during pneumonia outbreaks. Paradoxically, multiple exposures of ewes to pneumonia were associated with diminished survival of their offspring during pneumonia outbreaks. Although there was support for waning and boosting immunity in ewes, models with consistent immunizing exposure were similarly supported. Translocated animals that had not previously been exposed were more likely to die of pneumonia than residents. These results suggest that pneumonia in bighorn sheep can lead to aging populations of immune adults with limited recruitment. Recovery is unlikely to be enhanced by translocating naïve healthy animals into or near populations infected with pneumonia pathogens.     

Effects of reproduction,genotype and anthelmintic treatment of ewes on Ostertagia spp. populations

Donald A. D., Morley F. H. W., Waller P. J., Axelsen A., Dobson R. J. and Donnelly J. R. 1982. Effects of reproduction, genotype and anthelmintic treatment of ewes on Ostertagia spp. populations. International Journal for Parasitology12: 403–411. Merino and Border Leicester × Merino (BL × M) ewes, nearly all of the same age and reared at the same site, were either unmated or mated to Border Leicester rams. Ewes of each genotype and reproductive status were untreated or were given a single pre-lambing drench with thiabendazole at 50 or 100 mg/kg a week before the start of lambing in spring on pastures at Canberra which had been contaminated during autumn and winter by adult sheep. The two genotypes grazed together within each combination of reproductive status and anthelmintic treatment which grazed separately. Thiabendazole was highly effective in removing both fourth stage larvae and adults of Ostertagia spp., the most abundant genus. Eight weeks after the pre-lambing drench lactating ewes carried larger Ostertagia spp. populations than did unmated ewes of both genotypes, but as a result of reinfection after treatment, differences between drenched and undrenched ewes were not significant. At this time lactating as well as unmated ewes harboured large populations of arrested early fourth stage larvae of Ostertagia spp. acquired during the last 8 weeks, showing that arrest of development is not prevented by lactation. There was strong evidence that some ingested larvae which became arrested in lactating ewes were rejected by unmated ewes. At all stages of the reproductive cycle studied, BL × M ewes were substantially more resistant to Ostertagia spp. infection than Merinos. No persistent benefits in parasite control or in animal production were detected from the pre-lambing drench.     

Determinants and consequences of age of primiparity in bighorn ewes

Because variation in age of first reproduction can have major effects on individual fitness and population dynamics, it is important to understand what maintains that variability. Although early primiparity is assumed to be costly, it is sometimes associated with high lifetime reproductive success. We used a long‐term study on bighorn sheep Ovis canadensis to determine what variables affect age at first reproduction, investigate the impact of primiparity on body resources and quantify the reproductive performance of primiparous ewes. We then examined the consequences of delayed primiparity on adult body mass, longevity and lifetime reproductive success. Environmental conditions during early development, body mass as a yearling, genotype and maternal effects affected age of primiparity. Primiparous ewes lost more mass in winter and gained less mass in summer than multiparous ewes. Small yearling ewes that postponed reproduction attained similar adult mass than heavy yearling ewes who reproduced at a younger age. Early primiparity did not reduce longevity and was positively associated with lifetime reproductive success. Starting to reproduce as soon as possible appears to maximize fitness of females. When early life conditions are unfavorable, however, delayed primiparity allows greater body growth and likely maximizes survival. The combination of a conservative reproductive strategy and maternal effects on age of primiparity may partly delay population recovery following density‐dependent declines.     

Acute alcohol exposure, acidemia or glutamine administration impacts amino acid homeostasis in ovine maternal and fetal plasma

Fetal alcohol syndrome (FAS) is a significant problem in human reproductive medicine. Maternal alcohol administration alters maternal amino acid homeostasis and results in acidemia in both mother and fetus, causing fetal growth restriction. We hypothesized that administration of glutamine, which increases renal ammoniagenesis to regulate acid–base balance, may provide an intervention strategy. This hypothesis was tested using sheep as an animal model. On day 115 of gestation, ewes were anesthetized and aseptic surgery was performed to insert catheters into the fetal abdominal aorta as well as the maternal abdominal aorta and vena cava. On day 128 of gestation, ewes received intravenous administration of saline, alcohol [1.75 g/kg body weight (BW)/h], a bolus of 30 mg glutamine/kg BW, alcohol + a bolus of 30 mg glutamine/kg BW, a bolus of 100 mg glutamine/kg BW, alcohol + a bolus of 100 mg glutamine/kg BW, or received CO₂ administration to induce acidemia independent of alcohol. Blood samples were obtained simultaneously from the mother and the fetus at times 0 and 60 min (the time of peak blood alcohol concentration) of the study. Administration of alcohol to pregnant ewes led to a reduction in concentrations of glutamine and related amino acids in plasma by 21–30 %. An acute administration of glutamine to ewes, concurrent with alcohol administration, improved the profile of most amino acids (including citrulline and arginine) in maternal and fetal plasma. We suggest that glutamine may have a protective effect against alcohol-induced metabolic disorders and FAS in the ovine model.     

Persistent changes in arterial blood gases in fetal sheep

Two anaesthetic protocols were compared using pregnant sheep. In both groups of animals, anaesthesia was induced using an intravenous (i.v.) injection of diazepam and ketamine. The ewes were then intubated for positive pressure ventilation using 0.8 L/min of nitrous oxide and 2 L/min oxygen with 1.1-1.8% halothane. If the ewe showed any signs of awakening, one of two protocols was followed. First, the halothane concentration was increased to 2-3% until the ewe was completely anaesthetized. Second, the halothane concentration was not altered, but the ewe was given doses of i.v. diazepam (0.1 mg/kg) and ketamine (1 mg/kg) until again completely anaesthetized. At the completion of surgery, maternal recovery was rapid and similar between the two groups. However, five days after surgery, the fetal arterial Po(2) and oxygen content of the fetuses receiving additional halothane (1.9 +/- 0.2 kPa and 4.4 +/- 1.0 mL/100 mL) were statistically significantly depressed when compared with the fetuses receiving additional diazepam and ketamine (2.9 +/- 0.1 kPa and 7.0 +/- 0.5 mL/100 mL). These results led us to conclude that certain anaesthetic protocols, in spite of good maternal recovery, can lead to deleterious effects upon the fetus that persist for at least five days after surgery.     

Population subdivision among desert bighorn sheep (Oviscanadensis) ewes revealed by mitochondrial DNA analysis

We used behavioural observations and mitochondrial DNA (mtDNA) sequence analysis to examine demographic and genetic structure within and among home-range groups of desert bighorn sheep (Oviscanadensis) ewes in the Peninsular Ranges of southern California, USA. We identified substantial genetic variation in the first 515 bp of the mtDNA control region and determined that seven haplotypes were distributed in a nonrandom fashion among these ewe subpopulations. Although a significant (P < 0.01) amount of mtDNA variation (33%) was partitioned among home-range groups, we did not find strong evidence for matrilineal substructuring within these groups. Based on analyses of molecular variance, and comparisons of behavioural associations and distances between centres of activity, we concluded that within a given home-range group, bighorn sheep ewes generally associate with other ewes based on their availability rather than their matrilineal relationships. Our results also supported the conclusion that multiple ewe subpopulations exist within the Peninsular Ranges, and that these subpopulations are the most basic demographic and genetic units.     

Do Reproductive Status and Lamb Gender Affect the Foraging Behavior of Bighorn Ewes?

In female ungulates lactation carries a high energetic cost and mothers often devote more care to sons than to daughters. The aims of this study were to determine whether lactating bighorn ewes have higher foraging time, bite rate or selectivity of forage than barren ewes and whether reproductive status affects migration patterns. Ewes with male lambs were predicted to spend more time foraging and to have a higher bite rate than ewes with female lambs. There were no differences in foraging behavior according to ewe reproductive status from April to August. In September-November lactating ewes had a higher bite rate and spent more time foraging than nonlactating ewes but lamb gender did not affect foraging behavior. Lactating ewes gained less weight than nonlactating ones until mid-August; from mid-August to late September mass gain was similar for both groups of ewes. Nonlactating ewes spent more time standing but reproductive status did not affect vigilance behavior or step rate while foraging. Ewes with lambs did not differ from nonlactating ewes in step rate. Pregnant ewes migrated earlier than barren ewes to alpine areas in spring. By so doing they abandoned areas with good quality forage presumably to give birth in areas safer from predation. All ewes spent most of the summer in the alpine range but nonlactating ewes returned to the winter range earlier than lactating ewes, probably to profit from the abundant forage there.     

Fecal counts of lungworm larvae and reproductive effort in bighorn sheep, Ovis canadensis

Because parasite resistance and reproduction require metabolic resources, life-history models predict a tradeoff between current reproduction and parasite load. These tradeoffs have been widely studied in birds, but few studies have been conducted on mammals. We monitored lungworm ( Protostrongylus spp.) larvae counts in bighorn sheep ( Ovis canadensis ) over four years to examine how individual differences in fecal output of lungworm larvae (LPG) by yearlings and adults were affected by season, sex, body mass, age and reproductive effort. We also compared lamb mass at six months and LPG. Overall, we found that LPG varies seasonally, peaking in females prior to lambing and in males during the rut. Age had no effect on LPG for either sex. During autumn, we found no effect of age or mass on LPG for sheep one year and older. Lamb body size or sex did not affect LPG. Females that weaned a lamb had higher counts than females that did not produce a lamb or females whose lamb died during summer. For rams, social rank and testosterone levels were not related to LPG but LPG increased with time spent searching for estrous ewes during the rut. Our results suggest a tradeoff between parasite resistance and reproductive effort in bighorn sheep of both sexes.     

Gender-related differences in diazepam effects on performance

In order to investigate the possible differences of diazepam effects in the two sexes, two placebo-controlled double-blind studies were conducted on healthy volunteer students. In one study the subjects received diazepam 10 mg alone or combined with 0.5 g/kg of alcohol in a parallel group design; in the other 0.2 mg/kg of diazepam or placebo were given in a cross-over manner. In both trials diazepam impaired the psychomotor skills of women more than men. The difference was similar in tasks measuring cognitive (digit symbol substitution), motor (balance of extraocular muscles) and sensory (critical flicker fusion) performances. Tapping speed was affected to a similar degree in both genders. Diazepam 10 mg did not cause impairment of body balance, a parameter sensitive to alcohol. The combined effect of diazepam and alcohol was of similar magnitude in both sexes in all objective tests. Subjectively the women felt themselves clumsier than did the men. The calming effect was similar in both groups. The results suggest that while the performance of women may be more vulnerable than men to impairment by diazepam they also are aware of it. The difference of effects is of such magnitude that it may cause bias in experiments unless carefully balanced groups are used.     

Weaning Conflict in Desert and Mountain Bighorn Sheep (Ovis canadensis): An Ecological Interpretation

Field data on weaning behavior in bighorn sheep (Ovis canadensis) were collected from populations in British Columbia, southern California, and one transplanted from the original B.C. study site to eastern Oregon. These areas were designated mountain, desert, and transplant, respectively. Seasons that were energetically stressful to lactating ewes were predicted. Summers were implicated for desert ewes; winters for ewes in colder and more seasonal northern environments. Although the temporal distribution of milk available to lambs varied between the three study populations, ewes generally weaned their lambs prior to the onset of the predicted stressful periods. In the desert, weaning occurred more abruptly than it did in the two more northern populations. The evolution of weaning strategies is discussed in subspecies of bighorn sheep based on proximate and ultimate factors.     

Reproductive characteristics of Chinese Hu sheep

The reproductive characteristics of Chinese Hu sheep are reviewed. Male and female lambs reached puberty at a mean age of 120 days and 180 days, respectively, with an average body weight of 18.54 ± 1.48 kg (n = 31) and 22.75 ± 1.98 kg (n = 24). Adult male and female sheep reproduced throughout the year. On average, ewes lambed 1.65 times per year (n = 1482). The mean ovulation rate of adult ewes was over 2.2 (n = 46). The average litter size of 150863 ewes raised under extensive conditions was 2.06. The early sexual maturity of male and female lambs was respectively related to high concentrations of testosterone and 17β-E₂ in blood plasma in the earlier postnatal period. The mechanism of high fecundity is not known.     

Diazepam and propofol used as anesthetics during open-heart surgery do not cause chromosomal aberrations in peripheral blood lymphocytes

Diazepam is a benzodiazepine with anticonvulsant, anxiolytic, sedative and muscle-relaxing properties. Many aspects of its toxicity have been investigated, including genotoxic and carcinogenic effects in various model systems. However, it is still unclear whether diazepam is in fact a genotoxic agent. Propofol is a rapid-onset, short-acting intravenous anesthetic agent. It is used widely for the induction and maintenance of anesthesia as well as for long-term sedation in intensive care units. There is limited information in the literature on its genotoxic effects. Both drugs are commonly used as anesthetic in patients undergoing open-heart surgery. Therefore, we investigated the possible genotoxic effects of propofol and diazepam in those patients, using a chromosomal aberration (CA) assay. Peripheral blood samples were collected from 45 patients before induction of anesthesia and at the end of the anesthesia with diazepam or propofol. In Group I (n=24), anesthesia was induced with 0.2 mg kg(-1) diazepam and 10 microg kg(-1) fentanyl. In Group II (n=21), anesthesia was induced with 1 mg kg(-1) propofol and 10 microg kg(-1) fentanyl. Pancuronium bromide (0.1 mg kg(-1)) was administered for skeletal muscle relaxation in both groups. Anesthesia was maintained by diazepam administration at 5 mg kg(-1) in Group I or by continuous propofol administration at 2-4 mg (kg h)(-1) in Group II. All patients received 0.02 mg kg(-1) pancuronium and 5 microg kg(-1) fentanyl boluses at 30-40 min intervals for anesthesia maintenance. Body temperature was controlled during bypass in the two groups. We found that the mean frequency of CAs in both groups before and at the end of the anesthesia were not statistically significantly different. Our analysis also indicated that age, smoking habit and gender were not confounding factors. In conclusion, our results indicate that diazepam and propofol do not exert genotoxic effects in blood cells during open-heart surgery.     

Relationships among fecal lungworm loads, fecal glucocorticoid metabolites, and lamb recruitment in free-ranging Rocky Mountain bighorn sheep

Most wild Rocky Mountain big-horn sheep (Ovis canadensis canadensis) in northern latitudes are infected with lungworms. Indirect effects of lungworms on bighorn sheep are unknown, but high pulmonary burdens might increase stress (i.e., elevated glucocorticoid levels), and chronic stress could in turn decrease fitness. We hypothesized that high lungworm burdens in Rocky Mountain bighorn ewes increase stress, thereby increasing lamb mortality. To test our hypothesis, one subherd of bighorn sheep in Custer State Park, South Dakota was provided a free-choice loose mineral mix containing the anthelmintic fenbendazole every six weeks from March 1999 to August 2000 to eliminate lungworms; another subherd served as the control. Daily, individually marked ewes were located telemetrically from the ground and uniquely marked animals were observed until they defecated. After the herd moved from the area, fecal samples were collected and stored at -23 C. A consistent number of samples per season per herd (x-=16.56+/-3.99 samples) were collected. Fecal larval lungworm levels (LPG) in the treatment subherd were lower than levels in the control subherd; however, there was no difference in fecal glucocorticoid metabolite (FGM) levels between the two subherds. Fecal glucocorticoid metabolite levels varied by season in both subherds, with levels in winter lower than during the other three seasons. Lamb:ewe ratios were not different between the control and treatment subherds at the end of summer 1999. In contrast, the treatment group had a lower lamb:ewe ratio at the end of summer 2000 despite having lower LPG. However, this result was attributed to lower ewe production, not lower lamb survival. The LPG levels were not correlated with FGM concentrations; instead, FGM levels might reflect normal seasonal patterns. Other factors, including contagious ecthyma, were more important for determining lamb mortality than LPG and FGM levels during our study. We suggest further experimental work over a longer duration to address these relationships.     

Consistency of temperament in bighorn ewes and correlates with behaviour and life history

Individual differences in temperament may affect how animals react to novel situations, avoid predation, invest in reproduction and behave in a variety of social contexts. Little information is available, however, about individual differences in temperament for wild animals. For bighorn sheep, Ovis canadensis, ewes captured as part of a long-term study, we compared behaviour during handling to behaviour in the field and reproductive history. We considered 'bold' ewes those that were frequently trapped during the summer, and assigned to each ewe a docility index based on her behaviour during handling. Measurements of temperament for the same individual at different captures were highly consistent. Temperament was not affected by reproductive status or age, nor was it related to body mass. Correlations between behaviour at the trap and in the field were weak and mostly nonsignificant, suggesting that temperament is domain specific rather than domain general. Bold ewes tended to start reproducing earlier and have higher weaning success than shy ewes. Variability in temperamental traits in the study population could be maintained by life-history trade-offs and by yearly changes in selective pressures. Copyright 2000 The Association for the Study of Animal Behaviour.     

Mechanism of the effect of diazepam on acetylcholine concentration in mouse brain

Intraperitoneal injection of diazepam (20 mg/kg) caused an increase of free and bound acetylcholine in the mouse brain. Diazepam pretreatment (20 mg/kg) increased galanthamine anticholinesterase activity and its toxicity; as to physostigmine, armine, and paraoxon toxicity--it remained unchanged. It is supposed that diazepam blocks the release of aetylcholine from the cholinergic nerve terminals which should result in a decrease of functional acetylcholine concentrations in the synapse.     

Diazepam and pentobarbital dependence in the rat

Diazepam (100–133 mg/kg/day), administered chronically through a gastric fistula, and pentobarbital (ca 692 mg/kg/day), administered chronically in food, were studied for their dependence producing properties in Sprague-Dawley female rats. The diazepam abstinence syndrome was apparent 10 to 20 hours after withdrawal, persisted for over 60 hours and consisted of poker tail, explosive awakenings, digging in sawdust, jerks, tremors, wet dog shakes, hostility, decreased food and water consumption and weight loss. The pentobarbital abstinence syndrome came on rapidly peaking within 10 hours and was largely over by 16 hours. The pentobarbital abstinence syndrome differed from diazepam's by the presence of grand mal, clonic and atypical convulsions. Diazepam completely and in a dose related way suppressed the diazepam abstinence syndrome. Similarly pentobarbital suppressed the pentobarbital abstinence syndrome. The signs which could be suppressed in a dose related manner were different for the diazepam and pentobarbital abstinence syndromes. Diazepam only partially suppressed the pentobarbital abstinence syndrome and pentobarbital only partially suppressed the diazepam abstinence syndrome. These data indicate that diazepam and pentobarbital produce different types of dependencies in the rat and are not equivalent in suppressing signs of abstinence.     

Modification of the anticonvulsant efficacy of diazepam by Ro-15-1788 in the kindled amygdaloid seizure model

The effects of various doses of diazepam and the new central benzodiazepine antagonist Ro-15-1788 were investigated in fully amygdaloid kindled rats. Diazepam had a pronounced dose-dependent anticonvulsant effect in this model. Ro-15-1788 dose-dependently reduced the behavioral ranks of the elicited kindled seizures to a maximum of 60% of control without consistently modifying the afterdischarge duration. No prestimulation convulsant effects were seen with Ro-15-1788. When 2 mg/kg i.p. of Ro-15-1788 was given after various doses of diazepam, the prestimulation sedation and ataxia anticonvulsant effects of diazepam (0.5-2.0 mg/kg) were attenuated by treatment with 2 mg/kg dose of Ro-15-1788. At the low dose of diazepam (0.25 mg/kg), increased reduction of behavioral rank and after discharge duration was seen after the 2 mg/kg dose of Ro-15-1788. Thus, Ro-15-1788 appears not to have proconvulsant properties in the kindled amygdaloid seizure model. Further, Ro-15-1788 appears to have some anticonvulsant properties of its own. Mixed agonist and antagonist effects were seen with Ro-15-1788 when given after various doses of diazepam in this model.     

Buspirone: correlation of its anxiolytic and electroencephalographic effects

The influence of diazepam (1 and 5 mg/kg, i. p.) and buspirone (5 and 10 mg/kg) on the Fourier's spectral EEG power of sensomotor cortex and a conflict behavior in freely moving rats were studied. Diazepam (1 mg/kg) and buspirone (5 mg/kg) produced slowing of EEG theta-activity. Large doses of diazepam and buspirone produced different and multiple EEG effects. To 15-1788 (10 mg/kg) completely antagonized all the effects of diazepam (5 mg/kg). The authors discuss possible mutual relations between the influence on EEG and anxiolytic effect of these tranquilizers.