Morphological characterization of ejaculated cynomolgus monkey (Macaca fascicularis) sperm

The aim of this study was to give reference values for the frequency of morphological sperm abnormalities present in the semen from nonexperimental cynomolgus monkeys as well as for the dimensions of sperm heads. Spermatozoa from the liquid portion of electroejaculates from 14 cynomolgus monkeys were air‐dried as smears, fixed, and stained with Harris's Haematoxylin and subjected to visual analysis of morphology and computer‐aided analysis of ten morphometric variables. The majority (83%) of sperm were morphologically normal. Tail defects were the most common (11%), and showed the highest variation between individuals, the values ranging between 4 and 23%. Head abnormalities consisted of large, tapering, and amorphous forms but were not frequent (0.4%), the values ranging between 0 and 1.3%. Midpiece imperfections were found in all the individuals; the mean percentage was 5%, and the range varied between 3 and 9%. Tail plus midpiece was the only multiple abnormality observed, with a mean value of 1.5% and a range between 0 and 8%. The majority of these double defects consisted of a coiled tail together with a coiled midpiece. Mean values for the morphometric parameters characterizing sperm heads were as follows: area 17.2 μm², perimeter 15.2 μm, length 5.8 μm, width 4.0 μm, L/W ratio 1.5, gray‐level 98, ellipticity 0.2, first shape factor 0.9, second shape factor 1.4, and third shape factor 1.1. Overall coefficients of variation for the majority of parameters were below 7%, showing the great homogeneity in the dimensions of cynomolgus sperm heads. Most useful parameters for sperm characterization, according to their low variability, were perimeter, length, width, L/W ratio, and shape factors. Differences in these parameters were, however, observed between monkeys. Am. J. Primatol. 47:105–115, 1999. © 1999 Wiley‐Liss, Inc.     

Avian uterine fluid proteome: Exosomes and biological processes potentially involved in sperm survival

Uterine fluid is an aqueous milieu to which sperm are exposed during their storage and ascent. In this study, a bottom‐up proteomic strategy and bioinformatic analysis of hen uterine fluid was performed to improve the understanding of this fluid and its potential role in sperm survival mechanisms. The proteomic data were submitted to ProteomeXchange. Among the 913 proteins identified, 160 are known to be secreted and 640 are referenced in exosomes databases. We isolated exosomes from the avian uterine fluid, analyzed them using electron microscopy, and targeted several exosomes markers (ANXA1/2/4/5, VCP, HSP90A, HSPA8, PARK7, and MDH1) using immunoblotting. Electron microscopy and immunohistochemistry were also used to analyze uterovaginal junctions for the exosomal proteins ANXA4, VCP, and PARK7. Exosomes were observed both at the surface epithelium and inside sperm storage tubules. Our data were compared with two previously published studies on proteomic of hen uterine fluid, and with one study describing the proteomic content of rooster seminal plasma and sperm. In conclusion, we demonstrated for the first time that avian uterine fluid contains exosomes. These may play a key role in preserving sperm functions within the female genital tract. Their presence in the sperm storage tubules may represent an important mechanism regarding interaction between the female genital tract and sperm.     

Take it or leave it: transport of tools for future use by long-tailed macaques (Macaca fascicularis)

Future planning has previously been considered unique to humans. However, certain animals can foresee some of their future needs. We tested long-tailed macaques (Macaca fascicularis) ability to transport and after a delay use a rake, in two experiments. Firstly, six rake tool-trained macaques failed to select and after a delay, transport the rake. Secondly, five of the macaques were shaped on transporting the rake for immediate rewards. Now, all of the macaques transported and used the rake after a delay of 5 min; one was successful after 10 min and even on a few trials after 20 min. The macaques failed to plan in the first experiment, potentially because they were unable to foresee the appropriate sequence of behaviours to solve the task. After shaping, they displayed flexibility by successfully transporting tools after delays. Remarkably, several macaques generalized this behaviour by attempting to use non-provided objects or their tails. Our results show which information and experience macaques need in order to display tool transport for future use.     

Effect of amino acids on cryopreservation of cynomolgus monkey (Macaca fascicularis) sperm

The effects of three amino acids (proline, glutamine, and glycine) added to the freezing medium Tes-Tris-egg yolk (TTE) for cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa were studied. This is the first report on the effects of amino acids on nonhuman primate sperm cryopreservation. The addition of 5mM proline, 10mM glutamine, and 10 or 20mM glycine each significantly improved post-thaw sperm motility and membrane and acrosome integrity compared with the control (TTE alone). However, a significant decrease in motility and membrane/acrosome integrity was observed when amino acid concentrations increased to 60mM for proline and glutamine, and 80 mM for glycine. The results suggest that adding a limited amount of amino acids to the freezing media is beneficial for freezing cynomolgus monkey sperm.     

Prevalence of glucose intolerance in free-ranging Macaca fascicularis of Mauritius

Oral glucose tolerance tests (OGTTs) were carried out on 30 free-ranging long-tailed macaques (Macaca fascicularis) on the island of Mauritius, following the suggestion that severe glucose intolerance and diabetes mellitus might be prevalent in this macaque population. OGTTs revealed no evidence of frank diabetes mellitus in the sample. However, 13% of individuals showed impaired glucose tolerance, with preserved insulin secretion, suggesting the presence of the target tissue resistance to insulin characteristic of human noninsulin-dependent diabetes mellitus (NIDDM). The macaques with impaired glucose tolerance were neither obese nor aged. Glucose levels at all time points of the OGTT in normal macaques in our free-ranging sample were lower than reported in captive populations, perhaps due to greater physical activity. Our observations demonstrate that a genetic predisposition to glucose intolerance does exist in M. fascicularis, and that this condition, well documented in laboratory macaques, is not simply an artifact of captivity.     

Determining acrosomal status of the cynomolgus monkey (Macaca fascicularis) sperm by fluorescence microscopy

The acrosome of Macaca fascicularis sperm cannot be distinguished by conventional light microscopy, so determining whether sperm are acrosome-intact or-reacted is difficult. We describe methods for labeling the acrosomal region of sperm with two different probes: fluoresceinated Pisum sativum agglutinin and anti-sperm antiserum. Acrosome-intact sperm are much more heavily labeled in the acrosomal region than are acrosome-reacted sperm, providing a simple means of differentiating the two types of sperm. The two probes detect similar numbers of acrosome-reacted sperm following treatment with the divalent cation ionophore, A23187.     

Sensory,motor, and postganglionic sympathetic neurons forming the ulnar and radial nerves of two macaque species: Macaca nemestrina and Macaca fascicularis

The motor, sensory, and postganglionic sympathetic neurons forming the left ulnar and right radial nerves of long-tailed macaques (Macaca fascicularis) and pigtailed macaques (Macaca nemestrina) were localized by the horseradish peroxidase method of tracing neuronal connections. The ulnar and radial motoneurons formed a longitudinal column of variable extent in the lateral part of the ventral horn. In most animals, the ulnar motoneurons extended between the caudal ends of the C7 and T1 segments; the radial motoneurons extended between the rostral level of the C4 and the middle part of T1 segments. Although there were areas of overlap in the spinal distribution of ulnar and radial motoneurons, the ulnar motoneurons were located more dorsally and dorsolaterally than were the radial motoneurons. In most animals, labelled sensory neurons whose axons run with the ulnar nerve occurred in the C8–T4 dorsal root ganglia, and those whose axons run with the radial nerve occurred in the C5–T3 ganglia. The radial sympathetic neurons were distributed in stellate through T7 paravertebral sympathetic ganglia, and the ulnar sympathetic neurons were distributed in stellate through T4 paravertebral sympathetic ganglia. Though the motor, sensory, and sympathetic neurons forming the ulnar and radial nerves had wide segmental distributions, all showed peak frequencies in two segments. The cross-sectional areas of the motor, sensory, and postganglionic sympathetic neurons forming the radial and ulnar nerves were measured in the animal that showed the greatest amount of labelling for each nerve. The ulnar and radial motoneurons had a similar range of sizes, with cross-sectional areas between 120 and 2,160 μm². Most were smaller than 900 μm². The sensory neurons forming the ulnar and radial nerves also displayed a similar range of sizes, measuring between 120 and 3,360 μm² in cross-sectional area. Most neurons measured between 201 and 800 μm². The ulnar sympathetic neurons measured between 120 and 840 μm², and the radial neurons between 120 and 2,120 μm². In both cases, most neurons measured between 120 and 600 μm². The mean cross-sectional area for the radial sympathetic neurons was, however, larger than that for the ulnar sympathetic neurons.     

Latitudinal variation in cranial dimorphism in Macaca fascicularis

This study examines latitudinal and insular variation in the expression of sexual dimorphism in cranial length in three geographical groupings of Macaca fascicularis. In addition, the relationship between cranial length dimorphism (CLD) and sex‐specific size is examined. The results of the study identified a significant relationship between CLD and latitude for only one of the three geographic groupings. Sex‐specific relationships between cranial length and CLD were detected. The pattern of these relationships varied by geographic grouping. This study is important because it demonstrates that despite very similar levels of CLD in a single primate species, there exists important geographic variability in the correlates of that dimorphism. I suggest that geographically varying ecological factors may influence sex‐specific natural selection and the intensity of CLD in M. fascicularis. Gaining a better understanding of this geographical variability will require that future research examines morphological variation, including CLD, within its corresponding ecological and social contexts. Such research should be comparative, and incorporate multiple geographically separated populations with disparate environmental settings. Am. J. Primatol. 72:152–160, 2010. © 2009 Wiley‐Liss, Inc.     

Prenatal growth in the cynomolgus and rhesus macaque (Macaca fascicularis and Macaca mulatta): A comparison by ultrasonography

The rhesus monkey has played an important role in the history of reproductive research. Because of limitations on the exportation and availability of this species, several other species of nonhuman primates have been considered as alternate models. Among them is the crab-eating, or cynomolgus, macaque (Macaca fascicularis), which displays similarities in developmental, reproductive, and physiological parameters. The use of both species of macaques for pregnancy-related studies necessitates the assessment of differences in growth and development through gestation. Observations during the embryonic and fetal periods in both species have been compared with diagnostic ultrasound. Results indicate no significant differences in size during the embryonic and early fetal periods, but a greater acceleration of growth in the rhesus begins at approximately 100–110 gestational days (GD). Analysis of embryonic and fetal heart rates indicate no differences between the two species. Normal predictive values for a variety of growth parameters including gestational sac, greatest length, biparietal diameter, and femur length have boon calculated by multiple regression analysis. These charts have proven useful for confirming the gestational age after timed matings and for predicting the age of animals for which the conception date is not known.     

Movement characteristics of ejaculated sperm from cynomolgus monkeys (Macaca fascicularis) analyzed by manual and automated computerized image analysis

Sperm motility is an important indicator of male reproductive function. An automated computerized system was used to measure the movement characteristics of cynomolgus monkey sperm. Swimming velocities were in good agreement with data derived from tracking sperm heads manually with a digitizer, but sperm counting by the system was erroneous. In some ejaculates, there were two subpopulations of sperm with different curvilinear velocities, linearities of swim-paths, and lateral movements of the heads.     

Affiliation Between Aggressors and Third Parties Following Conflicts in Long-Tailed Macaques (Macaca fascicularis)

Studies on cercopithecine monkeys have shown that soon after an agonistic conflict, victims have increased rates of affiliation with the agressor—reconciliation—but not with other group members. Postconflict affiliation is thought to function to restore disturbed relationships and to reduce social tension. This study on a captive group of long-tailed macaques (Macaca fascicularis) is focused on postconflict affiliative behavior of the aggressor. Increased rates of contact between female aggressors and kin of the victim occurred, as well as between female aggressors and their own kin. Furthermore, there were increased rates of contact between aggressors—males and females—and other group members. The increase in contacts with the victim's kin was selective, i.e., it could not be ascribed to the increased contact tendency with group members in general, and was not a side effect of the aggressor's proximity to the victim due to reconciliation. The increase in contacts with own kin was not selective. The fact that male aggressors do not have increased postconflict contacts with their kin or with kin of the victim is in agreement with the notion that males are less integrated in the nepotistic matrilineal network than females are. The fact that studies by others that focused on the victim evidence no increase in postconflict contacts with kin of the opponent or with other group members may be explained by the aggressor's larger influence over the postconflict situation: to reduce social tension, it might be more effective to affiliate with the aggressor than with the victim. Our findings emphasize that conflicts influence the behavior of other monkeys besides the direct contestants and, thus, indicate that the disturbance of social homeostasis is a matter of concern for all group members.     

Influence of estradiol on cortisol secretion in ovariectomized cynomolgus macaques (Macaca fascicularis)

In an investigation of cortisol secretion in fully mature, ovariectomized cynomolgus monkeys (Macaca fascicularis), we compared monkeys that were given either placebo (OVX, n = 26) or 17beta estradiol (E(2 )) (EST, n = 26) in a daily oral dose. Serum cortisol concentrations were measured prior to the experimental manipulation and 3, 6, 9, and 12 months following initiation of treatment. Pretreatment cortisol values did not differ between groups. Assessment of the treatment period values revealed that cortisol concentrations were significantly higher ( approximately 10%) in the EST than in the OVX monkeys. Cortisol also varied significantly across periods of sampling. This time-dependent variation was attributable to elevations in months 6 and 9 (when daylight was generally long), relative to months 3 and 12 (when daylight was relatively short). The modest stimulatory effect of estrogen on corticosteroid production observed in this study is consistent with what has been seen in women, and contrasts with the more robust effects observed in New World monkeys. The possible relationship between season and cortisol secretion observed here has not been previously described in monkeys.     

Stone-tool usage by Thai long-tailed macaques (Macaca fascicularis)

In January and March of 2005, we conducted surveys of long-tailed macaques at Piak Nam Yai Island, Laem Son National Park (9 degrees N 34-35', 98 degrees E 28'), Ranong Province, situated in southern Thailand. Two of the three troops of long-tailed macaques found on the island were observed using axe-shaped stones to crack rock oysters, detached gastropods (Thais tissoti, Petit, 1852), bivalves (Gafrarium divaricatum, Gmelin, 1791), and swimming crabs (Thalamita danae, Stimpson, 1858). They smashed the shells with stones that were held in either the left or right hand, while using the opposite hand to gather the oyster meat. Some monkeys used both hands to handle the stones. According to Matsuzawa's 1996 hierarchical classification of tool usage (levels 0-3), the tool usage by Thai long-tailed macaques could be characterized as either level 1 (cracking rock oysters with stones) or level 2 (cracking drifting mollusks and crabs with stones by placing them on a rock). Our discovery of stone-tool usage by Thai long-tailed macaques provides a new point of reference for discussions regarding the evolution of tool usage and the material culture of primates.     

Morphology and somatometric growth of long-tailed macaques (Macaca fascicularis fascicularis) in Singapore

Crab-eating, or long-tailed, macaques [ Macaca fascicularis (Raffles, 1821)] have been studied extensively throughout their distribution in South and South-east Asia. Despite this extensive body of research, the island population of long-tailed macaques from Singapore remains virtually undescribed. In the present study, we compare the morphometric variability and patterns of growth observed in a population sample from Singapore with a composite sample from Thailand, north of the Isthmus of Kra. The results of our analyses indicate that there are statistically significant differences between the two populations in adult size and shape. For both males and females, the Singapore population is smaller than the Thai population. Relative to body length, the Singapore macaques exhibit significantly longer tails, and, relative to cranial length, they exhibit significantly more narrow faces than the Thai macaques. Although levels of sexual dimorphism for most morphometric traits are very similar, indicating similar levels of male–male competition for females, the Singapore males exhibit a significantly larger testicular volume relative to body weight, suggestive of an alternative male reproductive strategy. In addition to adult somatometric size and shape, comparisons of growth patterns relative to age and body size reveal significant differences between the two population samples. Combined, these results suggest either that statistically significant differences in adult morphology and patterns of growth can occur in presumably reproductively cohesive subspecies, or the Singapore macaques may be taxonomically distinct.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 675–694.     

Identification and characterization of P31m, a novel sperm protein in Cynomolgus monkey (Macaca fascicularis)

We have previously described a hamster sperm glycoprotein, P26h, which is implicated in the cascade of events occurring during the interaction between mature spermatozoa and the oocyte's zona pellucida. The P26h is acquired on the acrosomal cap of the spermatozoon during its maturation arising within the epididymis. Lately, using a polyclonal antiserum raised against P26h, a 34 kDa protein, P34H, has been identified on the acrosomal cap of the human spermatozoon. The cloning and sequencing of the cDNA encoding P34H has revealed a 65% similarity between the P34H and P26h amino acid sequences. Considering that P26h shows total immunocontraceptive properties in the hamster, it is of relevant importance to have an animal model phylogenetically closer to the human. Using the Cynomolgus monkey, we searched for a protein autologous to the human P34H. A 31 kDa protein, the P31m, localized on the acrosomal cap of the monkey spermatozoon has been identified by a Western blot analysis and by immunohistochemical techniques using an anti-hamster P26h antiserum. Northern blot analysis showed increasing high levels of the P31m mRNA through the epididymis and at lower levels in the testis. In situ hybridization showed the presence of the P31m mRNA in the principal cells of the epididymis. The cloning and sequencing of the cDNA encoding the P31m showed a high homology of 97% identity between the P31m and P34H nucleotidic sequences. This study clearly demonstrates that the monkey P31m is the homologous protein of the hamster P26h and of the human P34H. Mol. Reprod. Dev. 59: 431-441, 2001.     

Influence of Female Copulation Calls on Male Sexual Behavior in Captive Macaca fascicularis

We investigated the function of copulation calls—vocalizations by females during mating—in captive groups of long-tailed macaques. We tested predictions of the contest-competition, sperm competition, synchronized orgasms, mate again, alpha-male notification and graded-signal hypotheses. We observed 371 copulations of 36 females wherein the presence or absence of a copulation call was clear. Females call equally often with different males and shortly after ejaculation. Copulation calls occurred equally with copulations with and without ejaculation. Calls did not incite disruptions of the mating. Following calls females mated again, more often than expected, with their mating partner. Both pregnant and fertile females uttered copulation calls. Two females conceived and mated mainly with the alpha male then. We conclude that copulation calls do not incite male contest competition for sexual access to females and that it is unlikely that calls synchronize male and female orgasms. Several hypotheses remain plausible, but not all predictions are borne out unequivocably. This alerts us to the possibility that the calls could have multiple beneficial effects; natural selection might strike a compromise among functions. Investigation of the mate again, sperm competition and alpha-male notification hypotheses, and of hypotheses not tested in our study concerning female breeding overlap and female-female agonism, is required.     

Population genetics of crab-eating macaques (Macaca fascicularis) on the island of mauritius

Protein polymorphisms of the crab-eating macaque (Macaca fascicularis) on the island of Mauritius in the southwest Indian Ocean were examined electrophoretically for 201 blood samples. All of the variant types detected were those found previously in the Asian populations. Genetic variability of the island population was estimated to be P_poly = 16% and H = 6.5%, which was lower than that of the Asian populations. A tendency that a small number of loci were highly polymorphic was observed. These results may reflect the establishment of the island population from a small number of introduced ancestors, or the existence of the bottleneck effect. For the origin of the Mauritian monkeys, comparison of electrophoretic variants suggested that they originated from the Malay Peninsula or the Greater Sunda Islands, especially from Java. Though the homozygous individuals exceeded in number as a whole, a significant subdivision was not detected in the population. © 1993 Wiley-Liss, Inc.     

Comparison of rectal and infrared thermometry for obtaining body temperature in cynomolgus macaques (Macaca fascicularis)

BACKGROUND: It would be clinically advantageous to develop a method of body temperature evaluation in cynomolgus macaques (Macaca fascicularis) that did not require sedation, restraint, surgical manipulation, or expensive equipment. METHODS: Body temperatures of 51 cynomolgus macaques were taken with rectal thermometry and non-contact infrared thermometry (NIFT) on the shoulder, face, abdomen, and axillary region. RESULTS AND CONCLUSIONS: Body temperature measurements from NIFT were statistically different (P < 0.0001) from rectal thermometry. In addition, there was greater between- and within-subject variability in values using NIFT. There was no correlation between any sites of the NIFT and rectal thermometry. It was concluded that NIFT was not a valid alternative to rectal thermometry in cynomolgus macaques.     

The human-ABO blood groups of free-ranging long-tailed macaques (Macaca fascicularis) and parapatric rhesus macaques (M. mulatta) in Thailand

BACKGROUND: Long-tailed and rhesus macaques are widely used in biomedical research; therefore, the known blood group is important. METHODS: The human-type ABO blood group was determined in wild or semi-wild long-tailed and rhesus macaques in Thailand. A total of 729 long-tailed and 160 rhesus macaques from 20 localities were temporarily caught. RESULTS: The frequency profiles of blood groups, calculated by averaging the frequency of each troop in long-tailed and rhesus macaques, were AB > O > B > A at 29.6%, 27.4%, 27.2%, and 15.8%, and B > AB > A > O at 39.6%, 33.4%, 18.2%, and 8.8%, respectively. Irrespective of locality, the frequencies were AB > O > B > A of 29.6%, 28.0%, 24.4%, and 18.0%, and AB > B > A > O of 37.5%, 28.7%, 26.9%, and 6.9%, respectively, for all long-tailed and rhesus macaques. The frequency profile of blood groups in Thai rhesus macaques was somewhat similar to that in the parapatric long-tailed macaques; however, it was different from other rhesus populations where only group B was detected. CONCLUSIONS: Our data support the hypothesis that Indochinese rhesus macaques are hybrids between rhesus and long-tailed macaques in the past.     

Early embryonal culture of the cynomolgus monkey (Macaca fascicularis)

Ovaries of ten female cynologus monkeys (Macaca fascicularis) were superstimulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). During this treatment, follicular development was monitored by ultrasonography. After three to four injections of PMSG, ovaries began to show an increase in their size. After seven to ten injections of PMSG and a hCG injection, a total of 183 oocytes (18.3±14.1/ animal, mean±SD) were recovered from these females by laparotomy. Five nulliparous females produced 24.2±18.2 oocytes per animal, and the remaining five parous females produced 12.5±5.46 oocytes per animal. In total, 133 (72.6%) of the 183 oocytes were classified morphologically as matured, and 130 of them were inseminated with cynomolgus monkey sperm for 18 hours in vitro. The second polar body was observed in 92 (71%) of the inseminated oocytes, and 54 (59%) of the 92 oocytes showed cleavage division at least once. When these oocytes were fertilized and cultured in serum-free media, they stopped their development at 4-cell stage. In contrast to this condition, about a half of fertilized oocytes in serum-free media were able to develop to morula stage 4 days after insemination by transferring them into serum-containing media after 2-cell stage.