Differential distribution of sperm subpopulations and incidence of pleiomorphisms in ejaculates of captive howling monkeys (Alouatta caraya).

The aim of this study was to develop an objective method to determine the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates of howling monkeys (Alouatta caraya) by using a combination of computerized analysis system (ASMA) and principal component analysis (PCA) methods. Ejaculates were collected by electroejaculation methods on a regular basis from five individuals maintained under identical captive environmental, nutritional, and management conditions. Each sperm head was measured for dimensional parameters (Area [A, (square micrometers)], Perimeter [P, (micrometers)], Length [L, (micrometers)], and Width [W, (micrometers)]) and shape-derived parameters (Ellipticity [(L/W)], Elongation [(L - W)/(L + W)], and Rugosity [(4лA/P (2))]). PCA revealed two principal components explaining more than the 96 % of the variance. Clustering methods and discriminant analyzes were performed and seven separate subpopulations were identified. There were differences (P < 0.001) in the distribution of the seven subpopulations as well as in the incidence of abnormal pleiomorphisms (58.6 %, 49.8 %, 35.1 %, 66.4 %, and 55.1 %, P < 0.05) among the five donors tested. Our results indicated that differences among individuals related to the incidence of pleiomorphisms, and sperm subpopulational structure was not related to the captivity conditions or the sperm collection method, since all individuals were studied under identical conditions. In conclusion, the combination of ASMA and PCA is a useful clinical diagnostic resource for detecting deficiencies in sperm morphology and sperm subpopulations in A. caraya ejaculates that could be used in ex situ conservation programs of threatened species in Alouatta genus or even other endangered neotropical primate species.

Hematological, hepatic, and renal evaluation in Aotus azarai infulatus.

BACKGROUND: Knowing the physiological ranges of certain hematological and biochemical parameters is necessary for understanding the alterations caused by pathogenic agents and establishing differences between states of health and disease. Hematological evaluations and clinical chemistry were performed on livers and kidneys from owl monkeys with different sexes and age ranges. METHODS: Forty-one healthy owl monkeys were evaluated (26 males and 15 females) in three different age groups (AG1=3months to 1 year old; AG2=2-6years old; AG3=over 7years old). The animals received physical exams, abdominal ultrasounds, and clinical laboratory tests. RESULTS AND CONCLUSIONS: Significant differences were observed in hematocrit and hemoglobin values between males and females. The levels of alkaline phosphatase in the tissues were significantly influenced by age. There was also a sex influence on blood urea nitrogen and creatinine levels. The results show that some hematological and clinical chemistry parameters in owl monkeys may be influenced by sex and age.

Effect of age and number of parturitions on uterine and ovarian variables in owl monkeys.

BACKGROUND: We aimed to evaluate the uterine and ovarian volumes of owl monkeys in different age groups with different numbers of live births and to analyze the interaction between both. METHODS: We performed pelvic ultrasound exams to compare the uterine measurements with weight, age (infant, juvenile, subadult, young adults, and adults) and the number of live births (nulliparous, primiparous, and multiparous) and to compare the ovarian measurements with weight and age. RESULTS AND CONCLUSIONS: The uterine volume (UV) was directly proportional to the number of parturitions, which was the most important factor in the uterine growth of adult females (P < 0.05). The body weight and age of the animals showed a high positive correlation with UV (r = 0.5354, r = 0.6489, P < 0.01), respectively. The volume of the ovaries grew in proportion to the age of the females (P < 0.05). Puberty was the period of greatest uterine and ovarian growth.

DNA sequencing of 13 cytokine gene fragments of Aotus infulatus and Saimiri sciureus, two non-human primate models for malaria.

Aotus and Saimiri are non-human primate models recommended by the World Health Organization for experimental studies in malaria, especially for vaccine pre-clinical trials. However, research using these primates is hindered by the lack of specific reagents to evaluate immune responses to infection or vaccination. As a step toward developing molecular tools for cytokine expression studies in these species, primer pairs for 18 cytokine gene fragments were designed based on human DNA sequences and used to amplify the corresponding genes in Aotus infulatus and Saimiri sciureus genomic DNA samples. IFNγ, TNFα, LTA, IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, CSF2 and TGFß2 gene fragments were amplified and sequenced. Primer pairs for IL8, IL17, IL18, IL27 and MIF failed to generate amplification products. When compared to the available corresponding human and non-human primate sequences, most--except IL3 and IL4--showed identity degrees above 90%. Small variations in sequence can help to explain the failure to amplify certain genes or the amplification only at lower annealing temperatures as compared to human DNA samples for several primer pairs. The sequences made available provide the basis for designing molecular tools such as primers for real time PCR specific for A. infulatus and/or S. sciureus. The nucleotide sequences reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers DQ985386 to DQ985389, DQ989356 to DQ989369, FJ89020 to FJ89024, and FJ89029.

Validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (Callithrix jacchus) sperm.

Simple, rapid and stable sperm evaluation methods which have been optimized for common marmoset (Callithrix jacchus) are critical for studies involving collection and evaluation of sperm in the field. This is particularly important for new species groups such as Callitrichidae where the sperm have been little studied. Of this family, C. jacchus is the best known, and has been chosen as a model species for other members of the genus Callithrix. The fundamental evaluation parameters for sperm of any species are viability and acrosomal status. Semen samples were collected by penile vibratory stimulation. To evaluate sperm plasma membrane integrity, Eosin-Nigrosin was tested here for the common marmoset sperm to be used under field conditions. Further, a non-fluorescent stain for acrosome, the "Simple" stain, developed for domestic and wild cats, was tested on common marmoset sperm. This was compared with a fluorescent staining, Fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA), routinely used and validated for common marmoset at the German Primate Centre to evaluate acrosomal integrity. Results obtained with the "Simple" stain showed a marked differentiation between sperm with intact and non-intact acrosome both with and without ionophore treatment and closely correlated with results obtained with FITC-PSA. Temperature had no effect on the results with the "Simple" stain and the complete processing is simple enough to be carried out under field conditions. These findings indicated that the "Simple" stain and Eosin-Nigrosin provide rapid and accurate results for C. jacchus sperm and that those methods can be reliably used as field tools for sperm evaluation for this species.

Morphological and Morphometric Description of Female Reproductive Tract of Sapajus apella (Capuchin monkey).

Forest destruction has progressively hampered the survival of many species, and this is why it is so important to study of the lives of primates in captivity. This study aimed to describe the morphological aspects of the female reproductive tract of Sapajus apella. We used five animals obtained from the National Primate Center, Ananindeua - PA. The ovaries were paired, compact and symmetrical and had a smooth surface. The uterine tubes were bilateral and convoluted in adult animals and straight in young individuals. The uterus was simple and located in the pelvic region. The vagina was a long structure due to the position of the uterus. The external genitalia were located in the urogenital perineum and consisted of dark pigmented labia majora and labia minora, a vaginal vestibule as long as the vagina and a well-developed clitoris. The results showed that the genitals of S. apella resemble those of other Neotropical primates.

Chromosome studies in Chiropotes satanas utahicki hershkovitz, 1985 (cebidae, platyrrhini): A comparison with Chiropotes satanas chiropotes.

Karyological characterizations of C. s. utahicki (2n = 54) and C. s. chiropotes (2n = 54) showed that these two subspecies are chromosomally very similar. In a single, isolated specimen of C s. utahicki, however, a derived, biarmed, chromosome 14 was found in the heterozygous condition. This variant chromosome was identical with pair 10 in C. s. chiropotes in which this chromosome type was apparently fixed. Chromosome differences between these subspecies might be transitional, leading to the establishment of two different karyomorphic populations derived from a once uniform karyotypic group that split into separate allopatric subspecies. © 1992 Wiley-Liss, Inc.

Collection and evaluation of semen from captive howler monkeys (Alouatta caraya).

Semen samples (n=58) were collected by electroejaculation from nine adult male howler monkeys (Alouatta caraya) between November 2000 and August 2001 at the National Primates Center, Ananindeua, Brazil. The ejaculates were free of coagulum. Mean (+/-S.D.) values were: volume, 0.09 +/- 0.05 ml; pH, 8.1 +/- 0.5; concentration 649.5 +/- 926.7 x 10(6) sperm/ml; progressive motility, 75.8 +/- 18.1%; forward progressive sperm motility (scale, 0-5), 3.5 +/- 1.0; live spermatozoa, 68.3 +/- 15.0%; primary defects, 9.6 +/- 4.5%; and secondary defects, 11.8 +/- 4.6%. There were high correlations between motility and live sperm (r = 0.91, P < 0.01), motility and forward progressive sperm motility (r = 0.84, P < 0.01) and between forward progressive sperm motility and live sperm (r = 0.78, P < 0.01). There were no alterations observed during clinical examinations and hematological analysis performed before and after semen collection. Therefore, the method was considered safe and efficient. It can be used for the evaluation of the breeding potential of male howler monkeys in captivity and for the establishment of new assisted reproductive technology (ART) for threatened species of neotropical primates.

Identification of sperm head subpopulations with defined pleiomorphic characteristics in ejaculates of captive Goeldi's monkeys (Callimico goeldii).

The aim of this study was to evaluate the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates from the vulnerable Goeldi's monkey (Callimico goeldii) by using a combination of computerized analysis system and Principal Component Analysis (PCA) methods. Each sperm head was measured for four primary spermatozoal head dimensional parameters (area [A (µm(2))], perimeter [P (µm)], length [L (µm)] and width [W (µm)]) and three head shape derived parameters (ellipticity [(L/W)], elongation [(L-W)/(L+W)] and rugosity [(4πA/P(2))]). Six separate subpopulations (SPs) were identified: SP1, constituted by very large, narrow and very elliptical spermatozoa (A=16.85±1.56µm(2), W=2.75±0.42µm and ellipticity=2.16±0.24); SP2, characterized by average sized, short, wide and round spermatozoa (A=15.00±1.92µm(2), L=5.06±0.49µm, W=3.51±0.31µm and ellipticity=1.44±0.15); SP3, represented by small, wide and slightly round spermatozoa (A=14.95±1.75µm(2), W=3.47±0.29µm and ellipticity=1.48±0.14); SP4 included very small, short and very round spermatozoa (A=14.15±2.38µm(2), L=4.90±0.57µm and elongation=0.18±0.05); SP5 consisted of average sized and slightly elliptical spermatozoa (A=15.14±1.72µm(2) and ellipticity=1.49±0.14); and SP6 included large and round spermatozoa (A=16.30±1.62µm(2) and elongation=0.19±0.04). There were differences in the sperm subpopulation distribution (P<0.001) among the five donors analyzed. In conclusion, the results of the current study confirmed that the use of computer sperm analysis methods combined with PCA cluster analyses are useful methods to identify, classify, and characterize different sperm head morphometric subpopulations in neotropical primates. Broadening our knowledge of C. goeldii sperm morphometric abnormalities as well as developing reliable techniques for sperm evaluation may be essential for ex situ conservation of this threatened species.

Chromosomal studies in Callicebus donacophilus pallescens, with classic and molecular cytogenetic approaches: multicolour FISH using human and Saguinus oedipus painting probes.

This paper presents the karyotype of Callicebus donacophilus pallescens for the first time. The analysis included G-, C-, NOR-banding techniques and FISH with chromosome painting probes from Saguinus oedipus and Homo sapiens. The results were compared with the karyotypes of Callicebus moloch donacophilus and C. moloch previously published. These three karyotypes display the same diploid number (2n = 50) but diverge about the number of biarmed and acrocentric chromosomes. The acrocentrics 14 and 15 from C. m. donacophilus and C. moloch have undergone an in-tandem fusion originating a large acrocentric (pair 10) in C. d. pallescens. The major submetacentric pair (pair 1) from C. d. donacophilus and C. moloch have undergone fission originating two acrocentric pairs in C. d. pallescens (pairs 15 and 22). Herein was evidence that, in spite of the high interspecific variation among Callicebus, most of the chromosomes remained conserved.

Immunization of Saimiri sciureus monkeys with Plasmodium falciparum merozoite surface protein-3 and glutamate-rich protein suggests that protection is related to antibody levels.

The immunogenicity and protective efficacy of various antigen-adjuvant formulations derived either from the merozoite-surface protein-3 (MSP-3) or the glutamate-rich protein (GLURP) of Plasmodium falciparum were evaluated in Saimiri sciureus monkeys. These proteins were selected for immunogenicity studies based primarily on their capacity of inducing an antibody-dependent cellular inhibition effect on parasite growth. Some of the S. sciureus monkeys immunized with MSP-3(212-380)-AS02 or GLURP(27-500)-alum were able to fully or partially control parasitaemia upon an experimental P. falciparum [Falciparum Uganda Palo Alto (FUP-SP) strain] blood-stage infection, and this protection was related to the prechallenge antibody titres induced. The data are indicative that MSP-3 and GLURP can induce protective immunity against an experimental P. falciparum infection using adjuvants that are acceptable for human use and this should trigger further studies with those new antigens.

Mechanical isolation of capuchin monkey (Cebus apella) preantral ovarian follicles

The aim of this study was to adapt a mechanical procedure for the isolation of intact preantral follicles from Cebus apella ovaries. The interval effect of serial sections of the tissue chopper was tested on a number of preantral follicles isolated from ovaries (n=6) of three C. apella females, two prepubertal and one adult. Ovaries were divided into four equal parts and fragmented with a tissue chopper, adjusted for serial sections at intervals of 250, 500, 750 and 1,000æm, respectively. Isolated follicles were counted in a Neubauer's chamber and classified as primordial, primary or secondary. The number (mean±SE) of preantral follicles isolated from 1/4 ovary varied from 68,330+17,590 (at the 1,000æm cut interval) to 300,830+111,460 (at the 500æm cut interval. The mean diameter of the isolated preantral follicles varied from 11.6æm to 27.8æm.