Incorporation of a GnRH agonist,leuprolide acetate,into regimens with exogenous gonadotropins to produce ovarian stimulation and ovulation in the nonpregnant squirrel monkey

This study was designed to measure the effects of variations in the length of pretreatment with a GnRH agonist, leuprolide acetate (LA), on subsequent follicular development and ovulation. The hypothesis was that the duration of LA suppression of pituitary function does not adversely affect ovarian response to standardized ovulation induction protocols in squirrel monkeys. The first phase determined the dose and duration of LA needed to achieve a hypogonadal state. One of two groups received daily subcutaneous injections of 50 μg of LA. The other received a single injection of 175 μg of a depot suspension of LA. Sera were assayed for estradiol (E2) and progesterone (P). E2 and P levels increased 2‐ to 5‐fold with peak levels on days 4 and 7, respectively. Suppression of steroid levels took 10 to 15 days in the LA‐treated group. Depot‐LA did not effectively suppress steroid production. After suppression, females receiving daily LA received five daily injections of hMG to stimulate follicular development. E2 and P increased in these animals. These results suggest that cycling squirrel monkeys have P‐secreting capacity throughout the cycle. This may explain how the squirrel monkey is able to accommodate both a short (4–5 day) luteal phase of their 9 day cycle and implantation from 5 to 7 days after ovulation. A second study compared exogenous follicle stimulating hormone (FSH) to endogenous gonadotropins released as a response to LA in ovulation induction. Steroid production and hCG‐induced ovulation were assessed. LA treatment was compared to a standard ovulation induction protocol by using a randomized cross‐over measures design. There were no differences in E2 and P levels in response to dosages of either LA or hMG. The ovulatory response following LA treatment was not significantly greater than that using FSH. The number of animals with unovulated, large follicles was greater on the FSH protocol (12/18) compared to the LA protocol (3/18). Thus, a single injection of a depot preparation of LA is sufficient to stimulate follicular development and ovulation when followed by an hCG injection. Based on this observation and the data on unovulated large follicles, it is suggested that the ovary responds more readily to endogenous gonadotropins released by LA than to exogenous FSH. Am. J. Primatol. 49:153–164, 1999. © 1999 Wiley‐Liss, Inc.     

Confirmation of ovulation and characterization of luteinizing hormone and progesterone secretory patterns in cycling,isosexually housed Bolivian squirrel monkeys (Saimiri boliviensis boliviensis)

In the female Bolivian squirrel monkey a much greater elevation of serum estradiol (E₂) was measured after mating than that observed in similary cycling monkeys that did not mate. This raised the possibility that cycling squirrel monkeys may not ovulate during nonmated cycles To test this hypothesis, we performed laparoscopies on nine isosexually housed, cycling monkeys to observe the ovaries after the luteinizing hormone (LH) surge, which was measured by mouse interstitial cell bioassay using LER 1909-2 as the standard. Single ovulatory stigmas were identified as well demarcated, red, punctate depressions at the center of dome-shaped elevations on the ovarian surface in eight monkeys, when laparoscopically examined 9-56 hr after the LH peak. One monkey examined laparoscopically prior to the LH surge had a large translucent cystic follicle, confirming the morphology of the mature prevulatory follicle. Mean progesterone (P) concentrations fell to a nadir 1 day prior to the LH surge and then began to rise on the LH surge. Peak P levels were found 2 days after the LH surge. In the ovulating animals in which periovulatory E₂ levels were measured, no value was greater than 800 pg/ml, indicating that the presence of follicular rupture was not sufficient to account for the elevated E₂ levels observed after mating. These data confirm ovulation and follicular rupture in the absence of mating and delineate the relationship between periovulatory LH, P, and E₂ secretory patterns in cycling squirrel monkeys.     

Androgen resistance in squirrel monkeys (Saimiri spp.)

The goal of this study was to understand the basis for high androgen levels in squirrel monkeys (Saimiri spp.). Mass spectrometry was used to analyze serum testosterone, androstenedione, and dihydrotestosterone of male squirrel monkeys during the nonbreeding (n = 7) and breeding (n = 10) seasons. All hormone levels were elevated compared with those of humans, even during the nonbreeding season; the highest levels occurred during the breeding season. The ratio of testosterone to dihydrotestosterone in squirrel monkeys is high during the breeding season compared to man. Squirrel monkeys may have high testosterone to compensate for inefficient metabolism to dihydrotestosterone. We also investigated whether squirrel monkeys have high androgens to compensate for low-activity androgen receptors (AR). The response to dihydrotestosterone in squirrel monkey cells transfected with AR and AR-responsive reporter plasmids was 4-fold, compared with 28-fold in human cells. This result was not due to overexpression of cellular FKBP51, which causes glucocorticoid and progestin resistance in squirrel monkeys, because overexpression of FKBP51 had no effect on dihydrotestosterone-stimulated reporter activity in a human fibroblast cell line. To test whether the inherently low levels of FKBP52 in squirrel monkeys contribute to androgen insensitivity, squirrel monkey cells were transfected with an AR expression plasmid, an AR-responsive reporter plasmid, and a plasmid expressing FKBP52. Expression of FKBP52 decreased the EC50 or increased the maximal response to dihydrotestosterone. Therefore, the high androgen levels in squirrel monkeys likely compensate for their relatively low 5 alpha-reductase activity during the breeding season and AR insensitivity resulting from low cellular levels of FKBP52.     

Gustatory responsiveness to monosodium glutamate and sodium chloride in four species of nonhuman primates

The taste responsiveness of six squirrel monkeys, five pigtail macaques, four olive baboons and four spider monkeys to monsodium glutamate (MSG) and to sodium chloride was assessed in two-bottle preference tests of brief duration (2 min). When given the choice between tap water and defined concentrations of the two tastants dissolved in tap water, the animals were found to significantly discriminate concentrations of MSG as low as 2 mM (spider monkeys and olive baboons), 50 mM (pigtail macaques) and 300 mM (squirrel monkeys) from the solvent. With sodium chloride, taste preference thresholds were found to be 1 mM (spider monkeys), 20 mM (pigtail macaques), 50 mM (olive baboons), and 200 mM (squirrel monkeys), respectively. Across-species comparisons of the degree of preference for MSG and sodium chloride displayed by the four primate species showed the same order of spider monkeys>olive baboons>pigtail macaques>squirrel monkeys. When presented with equimolar concentrations of different tastants, all four species preferred sucrose as well as a mixture of sucrose and sodium chloride over MSG, and--at least at one concentration--they preferred MSG over sodium chloride. The results support the assertion that the taste responsiveness of the four primate species to MSG and sodium chloride might reflect an evolutionary adaptation to their respective dietary habits.     

Vaccination of squirrel monkeys (Saimiri spp.) with nanoparticle-based Toxoplasma gondii antigens: new hope for captive susceptible species

Squirrel monkeys (Saimiri spp.), new world primates from South America, are very susceptible to toxoplasmosis. Numerous outbreaks of fatal toxoplasmosis in zoos have been identified around the world, resulting in acute respiratory distress and sudden death. To date, preventive hygiene measures or available treatments are not able to significantly reduce this mortality in zoos. Therefore, vaccination seems to be the best long-term solution to control acute toxoplasmosis. Recently, we developed a nasal vaccine composed of total extract of soluble proteins of Toxoplasma gondii associated with muco-adhesive maltodextrin-nanoparticles. The vaccine, which generated specific cellular immune responses, demonstrated efficacy against toxoplasmosis in murine and ovine experimental models. In collaboration with six French zoos, our vaccine was used as a last resort in 48 squirrel monkeys to prevent toxoplasmosis. The full protocol of vaccination includes two intranasal sprays followed by combined intranasal and s.c. administration. No local or systemic side-effects were observed irrespective of the route of administration. Blood samples were collected to study systemic humoral and cellular immune responses up to 1 year after the last vaccination. Vaccination induced a strong and lasting systemic cellular immune response mediated by specific IFN-γ secretion by peripheral blood mononuclear cells. Since the introduction of vaccination, no deaths of squirrel monkeys due to T. gondii has been observed for more than 4 years suggesting the promising usage of our vaccine. Moreover, to explain the high susceptibility of naive squirrel monkeys to toxoplasmosis, their innate immune sensors were investigated. It was observed that Toll-like and Nod-like receptors appear to be functional following T. gondii recognition suggesting that the extreme susceptibility to toxoplasmosis may not be linked to innate detection of the parasite.     

Second-order schedules of drug injection.

Key-press responding of squirrel monkeys produced intravenous injections of cocaine under two simple types of schedule. Under a fixed ratio schedule, every 30th response produced an injection; steady responding at high rates of over one per second were maintained during each fixed-ratio component. Under a fixed-interval schedule, the first response occurring after a fixed time of 5 min produced an injection; there was a pause at the start of each interval and then progressively increasing responding until cocaine was injected at the end of the interval. Both squirrel monkeys and rhesus monkeys also were studied under second-order schedules of drug injection. Under one type of second-order schedule, studies only in squirrel monkeys, completion of each fixed-interval component produced only a 2 sec light; completion of the 10th fixed-interval component produced the brief light and an intravenous injection of cocaine. Under a second type of second-order schedule, each fixed-ratio component completed during a fixed time interval (5 or 60 min) produced only a 2-sec light; the first fixed-ratio component completed after the interval of time elapsed produced the brief light and an intravenous (squirrel monkeys) or intramuscular (rhesus monkeys) injection of cocaine. Under both types of second-order schedules, repeated sequences of responding were maintained during each session and characteristic fixed-interval or fixed-ratio patterns of responding were controlled by the brief visual stimuli.     

Social grooming between squirrel monkeys and uakaris in a seminatural environment

Young squirrel monkeys (Saimiri sciureus) were reported grooming an adult female uakari (Cacajao calvus rubicundus) on four different occasions. Furthermore, the uakari was noted grooming two squirrel monkeys in separate instances. These observations took place in a seminatural rainforest (The Monkey Jungle; Goulds, Florida, U.S.A.) where provisions are provided. Some possible hypostheses tendered to account for this unusual behavior included (a) the unaverted interaction of food-seeking and fur-cleaning behavior, and (b) the compatibility of play-curiosity activities by squirrel monkeys with the uakaris' need for social contact.     

Ovarian stimulation of squirrel monkeys (Saimiri boliviensis boliviensis) using pregnant mare serum gonadotropin

The application of assisted reproductive technologies (ART) to nonhuman primates has created opportunities for improving reproductive management in breeding colonies, and for creation of new animal models by genetic modification. One impediment to the application of ART in Saimiri spp. has been the lack of an effective gonadotropin preparation for ovarian stimulation. Pregnant mare serum gonadotropin (PMSG) is inexpensive and readily available, but its repeated use in rhesus monkeys has been associated with induction of a refractory state. We have compared PMSG to recombinant human follicle stimulating hormone (rhFSH) for controlled ovarian stimulation in Bolivian squirrel monkeys. Groups of mature squirrel monkeys received rhFSH (75 IU daily) or PMSG (250 IU twice daily) by subcutaneous injection for 4 d during the breeding season (November to January) or nonbreeding season (March to September). Serum estradiol (E2) was measured daily. Follicular growth was monitored by abdominal ultrasound. During the breeding season, PMSG induced a higher E2 response than did rhFSH, with mean E2 levels being significantly higher within 3 d of stimulation. Superior follicular development in PMSG animals was confirmed by abdominal ultrasonography. During the nonbreeding season, PMSG elicited a similar increase in serum E2 levels despite the fact that basal serum E2 is typically low during the nonbreeding season. Repeated use of PMSG (< or = 3 cycles of administration) produced no attenuation of the E2 response. We conclude that PMSG is highly effective for repeated cycles of controlled ovulation stimulation in the squirrel monkey.     

Determinants of problem-solving success inSaimiri andCallicebus

The problem-solving behavior of titis and squirrel monkeys was compared on four tasks. Each task was presented at three levels of complexity, based on the number of responses required to obtain a food reward. At all levels, however, food could be obtained by performing a few relatively simple and generalized responses, such as grasping, lifting, or pulling, that were within the repertoire of each species. The principal measures of problem-solving efficiency were the number of trials on which the reward was obtained, and the time required to achieve it. On both measures squirrel monkeys were superior to titis. These differences were not attributable to appetite for food, availability of the requisite instrumental responses, or cognitive ability. The most important factor contributing to the superior achievement of squirrel monkeys was problem-solving “style,” as reflected in latency to approach the problem, tempo of activity, and vigor and variety of responses emitted. On problems where the relevance of these traits was minimal, the level of successful performance was similar in the two species. The importance of taking style into account in comparative assessments of primate problem-solving ability is emphasized by these results.     

Tonic immobility in the squirrel monkey (Saimiri sciureus)

Tonic immobility and several accompanying behavioral changes were examined in the squirrel monkey (Saimiri sciureus). It was found that either the occurrence of a loud noise immediately before induction or the presence of a stuffed Cooper's hawk increased the duration of immobility in some subjects. Subspecies differences in durations of immobility were also found. The number of inductions required to produce immobility in squirrel monkeys were unusually large, and were negatively correlated with duration of the response. Behavioral variables accompanying tonic immobility showed qualitative differences when compared to what is known concerning their relation to immobility in other species. The findings support the belief that tonic immobility evolved as a predator defense in squirrel monkeys or one of their ancestors, but suggest that it no longer plays an active role in their survival.     

Comparison of rectal and tympanic core body temperature measurement in adult Guyanese squirrel monkeys (Saimiri sciureus sciureus)

Background Measuring core body temperature in a manner that is safe for animals and veterinary personnel is an important part of a physical examination. For nonhuman primates, this can involve increased restraint, additional stress, as well as the use of anesthetics and their deleterious effects on body temperature measurements. The purpose of this study was to compare two non‐invasive methods of infrared tympanic thermometry to standard rectal thermometry in adult squirrel monkeys. Methods Tympanic temperatures were collected from 37 squirrel monkeys and compared to rectal temperatures using a human and veterinary infrared tympanic thermometer. Results Compared with rectal temperature measurements, the human tympanic thermometer readings were not significantly different, while the veterinary tympanic thermometer measurements were significantly higher (P < 0.05). There were no differences between sexes. Conclusions The tympanic thermometer designed for use in humans can be used in adult squirrel monkeys as an alternative to rectal thermometry for assessing core body temperature.     

Cortisol metabolism in the Bolivian squirrel monkey (Saimiri boliviensis boliviensis)

New World squirrel monkeys (Saimiri spp.) have high circulating cortisol levels but normal electrolytes and blood pressures. The goal of the present study was to gain insight into adaptive mechanisms used by Bolivian squirrel monkeys to minimize the effects of high cortisol on mineralocorticoid receptor (MR) activity and electrolyte and water balance. Aldosterone levels in serum from 10 squirrel monkeys were 17.7 +/- 3.4 ng/dl (normal range in humans, 4 to 31 ng/dl), suggesting that squirrel monkeys do not exhibit a compensatory increase in aldosterone. The squirrel monkey MR was cloned and expressed in COS-7 cells and found to have similar responsiveness to cortisol and aldosterone as human MR, suggesting that squirrel monkey MR is not inherently less responsive to cortisol. To determine whether altered metabolism of cortisol might contribute to MR protection in squirrel monkeys, serum and urinary cortisol and cortisone were measured, and a comprehensive urinary corticosteroid metabolite profile was performed in samples from anesthetized and awake squirrel monkeys. The levels of cortisone exceeded those of cortisol in serum and urine, suggesting increased peripheral 11beta-hydroxysteroid dehydrogenase 2 activity in squirrel monkeys. In addition, a significant fraction (approximately 20%) of total corticosteroids excreted in the urine of squirrel monkeys appeared as 6beta-hydroxycortisol, compared with that in man (1%). Therefore, changes in cortisol metabolism likely contribute to adaptive mechanisms used by Bolivian squirrel monkeys to minimize effects of high cortisol.     

Age differences and developmental trends in alarm peep responses by squirrel monkeys (Saimiri sciureus)

Alarm calls can code for different classes of predators or different types of predatory threat. Acoustic information can also encode the urgency of threat through variations in acoustic features within specific alarm call types. Squirrel monkeys (Saimiri sciureus) produce an alarm call, known as the alarm peep, in highly threatening situations. Infant squirrel monkeys appear to have an innate predisposition to respond to alarm peeps but require experience to associate alarm peeps with the appropriate type of predatory threat [Herzog & Hopf, American Journal of Primatology 7:99-106, 1984]. Little is known about age-related differences in the type or frequency of response to alarm peeps, or the development of alarm peep response in infants. The purpose of this study was to test experimentally the response strategies of different age classes of squirrel monkey to the playback of alarm peeps that were produced by infants, juveniles, or adults. Results suggest that infants, juveniles, and female subadults respond more frequently to alarm peeps than do adult females. Infant squirrel monkeys showed different behavioral strategies in response to alarm peeps as a function of age. Adult females differentiate between infant and adult alarm peeps by responding more frequently to the alarm peeps of adult females. These data demonstrate that squirrel monkeys use acoustic information to discern when to respond to the alarm peeps from conspecifics, and that infants gradually develop an adult-like response to alarm peeps over the first year of development.     

Postnatal changes of IgG and IgM levels in squirrel monkeys (Saimiri sciureus)

Serum IgG and IgM levels were measured in domestically bred squirrel monkeys (Saimiri sciureus) ranging in age from 0 days to 42 months, as well as in adult squirrel monkeys from the wild estimated to be 60 months or older. The results indicated that the transplacental transfer of IgG occurs in the squirrel monkey but the transferability is lower in the squirrel monkey than in the cynomolgus monkey. Immune response in the squirrel monkey occurs just after birth, as shown by IgM production.     

Taste preference thresholds for food-associated sugars in the squirrel monkey (Saimiri sciureus)

The purpose of this study was to determine taste preference thresholds for four different food-associated sugars inSaimiri sciureus. Using a two-bottle test three male squirrel monkeys were found to significantly prefer 10 mM sucrose, 40 mM fructose, 90 mM glucose, and 100 mM lactose over tap water. Presentation of suprathreshold sugar solutions led to marked concentration-dependent and monotonically increasing polydipsia. The results showed the squirrel monkey to prefer lower sugar concentrations compared to other non-human primates and thus support the assumption that this species may use sweetness as a criterion for food selection.     

Squirrel monkeys support replication of BK virus more efficiently than simian virus 40: an animal model for human BK virus infection

We performed experiments to test the suitability of squirrel monkeys (Saimiri sciureus) as an experimental model for BK virus (BKV) and simian virus 40 (SV40) infection. Four squirrel monkeys received intravenous inoculation with BKV Gardner strain, and six squirrel monkeys received intravenous inoculation with SV40 777 strain. Eight of 10 monkeys received immunosuppression therapy, namely, cyclophosphamide subcutaneously either before or both before and after viral inoculation. The presence of viral infection was assessed by quantitative real-time PCR amplification of viral DNA from blood, urine, and 10 tissues. We found that squirrel monkeys were susceptible to infection with BKV, with high viral copy number detected in blood and viral genome detected in all tissues examined. BKV genome was detected in urine from only one monkey, while three monkeys manifested focal interstitial nephritis. BKV T antigen was expressed in renal peritubular capillary endothelial cells. By contrast, SV40 was detected at very low copy numbers in only a few tissues and was not detected in blood. We conclude that the squirrel monkey is a suitable animal for studies of experimental BKV infection and may facilitate studies of viral entry, pathogenesis, and therapy.     

Phylogenomics of species from four genera of New World monkeys by flow sorting and reciprocal chromosome painting

Background

The taxonomic and phylogenetic relationships of New World monkeys (Platyrrhini) are difficult to distinguish on the basis of morphology and because diagnostic fossils are rare. Recently, molecular data have led to a radical revision of the traditional taxonomy and phylogeny of these primates. Here we examine new hypotheses of platyrrhine evolutionary relationships by reciprocal chromosome painting after chromosome flow sorting of species belonging to four genera of platyrrhines included in the Cebidae family: Callithrix argentata (silvered-marmoset), Cebuella pygmaea (pygmy marmoset), Callimico goeldii (Goeldi's marmoset) and Saimiri sciureus (squirrel monkey). This is the first report of reciprocal painting in marmosets.

Results

The paints made from chromosome flow sorting of the four platyrrhine monkeys provided from 42 to 45 hybridization signals on human metaphases. The reciprocal painting of monkey probes on human chromosomes revealed that 21 breakpoints are common to all four studied species. There are only three additional breakpoints. A breakpoint on human chromosome 13 was found in Callithrix argentata, Cebuella pygmaea and Callimico goeldii, but not in Saimiri sciureus. There are two additional breakpoints on human chromosome 5: one is specific to squirrel monkeys, and the other to Goeldi's marmoset.

Conclusion

The reciprocal painting results support the molecular genomic assemblage of Cebidae. We demonstrated that the five chromosome associations previously hypothesized to phylogenetically link tamarins and marmosets are homologous and represent derived chromosome rearrangements. Four of these derived homologous associations tightly nest Callimico goeldii with marmosets. One derived association 2/15 may place squirrel monkeys within the Cebidae assemblage. An apparently common breakpoint on chromosome 5q33 found in both Saimiri and Aotus nancymae could be evidence of a phylogenetic link between these species. Comparison with previous reports shows that many syntenic associations found in platyrrhines have the same breakpoints and are homologous, derived rearrangements showing that the New World monkeys are a closely related group of species. Our data support the hypothesis that the ancestral karyotype of the Platyrrhini has a diploid number of 2n = 54 and is almost identical to that found today in capuchin monkeys; congruent with a basal position of the Cebidae among platyrrhine families.

     

Serologic evidence of Encephalitozoon cuniculi infection in a colony of squirrel monkeys (Saimiri sciureus)

Five hundred seventeen serum samples obtained during 3 years from a collection of 250 squirrel monkeys were examined by indirect immunofluorescence and dot-ELISA for antibodies to Encephalitozoon cuniculi. One hundred seventy-nine monkeys were positive at least once and fifty six monkeys were positive three or more times. Older animals were more likely to be positive than young animals, but the proportion of serologically positive monkeys did not change appreciably over the 3 years. As judged by the serological evidence, infection with E. cuniculi is distributed widely in this collection of squirrel monkeys.     

Natural conceptual behavior in squirrel monkeys (saimiri sciureus): An experimental investigation

Natural conceptual discriminations have been tested in many different species, including pigeons and a variety of non-human primates. The ability of four male squirrel monkeys (Saimiri sciureus) to learn and use the natural concept ‘squirrel monkey’ was investigated in this study. After a training phase, subjects were presented with novel stimuli in transfer and test trials. All subjects performed at a rate significantly above chance on the first test trial (p<.001), indicating that squirrel monkeys can utilize natural concepts in the laboratory.     

Measures of body size and growth in rhesus and squirrel monkeys subjected to long-term dietary restriction

Although many studies have reported the robust effects of dietary restriction (DR) in retarding numerous aging processes in rodents, little is known about the outcomes of reducing caloric intake of a nutritious diet on aging in primates. Most primate studies have concerned the effects of malnutrition. We hypothesized that DR influences aging processes in primate species as it does in rodents. In the present study, 24 male rhesus (Macaca mulatta) monkeys (ages 0.6–5 years) and 25 male squirrel (Saimiri sp.) monkeys (ages 0.3–10 years) were provided diets formulated differently for each species but both fortified with vitamins and minerals (40% above recommended levels) as controls (approximating ad libitum levels) or experimentals (about 30% below the level of diet provided controls of comparable age and body weight). The results reported here concern the hypothesis that DR imposed during various developmental stages in these two primate species would affect morphometric parameters obtained at different occasions during the first 5 years of the study. Groups of older monkeys (rhesus: 18–25 years, n = 3; squirrel: 10–15 years, n = 4) were also included as controls for comparative purposes. Among groups of rhesus monkeys begun on DR prior to 6 years of age, growth in body weight and crown-rump length was reduced about 10–20% beginning after 1 year on the diet, with estimated food intake being reduced about 30–35% over this period. Measures of skin-fold thickness and various body circumference measures were also reduced in experimental groups of rhesus monkeys. In contrast, the DR regimen involving a different diet produced little impact on comparable measures in squirrel monkeys, with the estimated food intake being reduced only about 20–25% over this period. However evidence of divergence in some morphometric parameters in squirrel monkeys was beginning to emerge in young groups (<5 years(after 3 yers on the diet. © 1995 Wiley-Liss, Inc.