Morphological evaluation of adult domestic cat testicular biopsy after vitrification.

Testicular biopsies (9 mm3) from domestic cats (n = 10) submitted to orchiectomy were submitted to equilibrium vitrification in the presence of ethylene glycol (EG) alone or combined with dimethylsulfoxide (DMSO) as intracellular cryoprotectants, and sucrose or trehalose as extracellular cryoprotectants. The samples were vitrified with 40% EG or 20% EG + 20% DMSO, plus 0.1 M or 0.5 M of sucrose or trehalose. The study was divided into Step 1 and Step 2. In Step 1, intratubular cells (spermatogonia, spermatids, spermatocytes, and Sertoli cells) were quantified and classified as intact or degenerated (pyknotic and/or vacuolated cells). Cryodamage of seminiferous cords was determined by spermatogonia and Sertoli cell scoring of nuclei alterations, tubular basement membrane detachment, epithelium shrinkage, and tubular measures (total area, epithelium area, larger and smaller diameter, and height of the epithelium). In Step 2, Hoechst 33342 stain and propidium iodide (PI) fluorescent stain were used to assess the cell viability of the four best experimental groups in Step 1. The effect of treatments on all analyses was accessed using analysis of variance (ANOVA), and Fisher's post hoc test at P < 0.05 significance was considered. In Step 1, the mean percentage of spermatogonia and Sertoli cells morphological integrity did not show a difference when using both sugars at different concentrations, but their morphology was more affected when DMSO was used. EG use associated with 0.1 M of sucrose or trehalose positively affected spermatocyte and spermatid morphology, respectively. The larger diameter and epithelium height of seminiferous tubules were increased using DMSO plus 0.5 M sucrose and DMSO plus 0.1 M trehalose. The changes in spermatogonial/Sertoli nucleoli visualization were best scored in the EG groups, while the nuclei condensation was lower with sucrose. The basement membrane was satisfactorily preserved with 0.1 M sucrose. In Step 2, the percentage of cell viability was higher when EG plus 0.1 M sucrose was used. Therefore, DMSO's negative effect on the vitrification of testicular biopsies of adult domestic cats was evident. The EG plus 0.1 M of sucrose or trehalose associations are the most suitable CPAs to preserve the testicular histology structure of adult domestic cats in vitrification.

Kinetic vitrification: concepts and perspectives in animal sperm cryopreservation.

Sperm cryopreservation is an important tool for genetic diversity management programs and the conservation of endangered breeds and species. The most widely used method of sperm conservation is slow freezing, however, during the process, sperm cells suffer from cryoinjury, which reduces their viability and fertility rates. One of the alternatives to slow freezing is vitrification, that consist on rapid freezing, in which viable cells undergo glass-like solidification. This technology requires large concentrations of permeable cryoprotectants (P- CPA's) which increase the viscosity of the medium to prevent intracellular ice formation during cooling and warming, obtaining successful results in vitrification of oocytes and embryos. Unfortunately, this technology failed when applied to vitrification of sperm due to its higher sensitivity to increasing concentrations of P-CPAs. Alternatively, a technique termed 'kinetic sperm vitrification' has been used and consists in a technique of permeant cryoprotectant-free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen. Some of the advantages of kinetic vitrification are the speed of execution and no rate-controlled equipment required. This technique has been used successfully and with better results for motility in human (50-70% motility recovery), dog (42%), fish (82%) and donkey (21.7%). However, more studies are required to improve sperm viability after devitrification, especially when it comes to motility recovery. The objective of this review is to present the principles of kinetic vitrification, the main findings in the literature, and the perspectives for the utilization of this technique as a cryopreservation method.

Zika Virus Infection Damages the Testes in Pubertal Common Squirrel Monkeys (Saimiri collinsi).

During the Zika virus (ZIKV) outbreak and after evidence of its sexual transmission was obtained, concerns arose about the impact of the adverse effects of ZIKV infection on human fertility. In this study, we evaluated the clinical-laboratory aspects and testicular histopathological patterns of pubertal squirrel monkeys (Saimiri collinsi) infected with ZIKV, analyzing the effects at different stages of infection. The susceptibility of S. collinsi to ZIKV infection was confirmed by laboratory tests, which detected viremia (mean 1.63 × 106 RNA copies/µL) and IgM antibody induction. Reduced fecal testosterone levels, severe testicular atrophy and prolonged orchitis were observed throughout the experiment by ultrasound. At 21 dpi, testicular damage associated with ZIKV was confirmed by histopathological and immunohistochemical (IHC) analyses. Tubular retraction, the degeneration and necrosis of somatic and germ cells in the seminiferous tubules, the proliferation of interstitial cells and an inflammatory infiltrate were observed. ZIKV antigen was identified in the same cells where tissue injuries were observed. In conclusion, squirrel monkeys were found to be susceptible to the Asian variant of ZIKV, and this model enabled the identification of multifocal lesions in the seminiferous tubules of the infected group evaluated. These findings may suggest an impact of ZIKV infection on male fertility.

Seasonal variations in gonadosomatic index and seminal quality of squirrel monkeys (Saimiri collinsi).

BACKGROUND: We hypothesized that seasonal variations in testicular biometry in Saimiri collinsi are influenced by body mass and that testicular enlargement is related to improved seminal quality. Therefore, the gonadosomatic index (GSI) can be a tool to determine reproductive seasons and the predictors of seminal quality. METHODS: Body mass, testicular biometry, and semen samples were obtained from eight adult males monthly and evaluated. RESULTS: In the non-fattening/non-reproductive season, the body mass and GSI decreased and increased, respectively, in the fattening. A positive correlation was only observed between the GSI and seminal volume. During fattening, high seminal volume and normal morphology were observed, but sperm concentrations were low. CONCLUSION: The GSI cannot be used as a predictor of seminal quality, but selective pressure to improve seminal quality may increase the chances of fertilization at fattening. The GSI provides new information on seasonality and can be used to identify reproductive seasons in squirrel monkeys.

Ultrasonography of the neotropical primate female reproductive system.

The Neotropical (e. g., Aotus sp., Callithrix jacchus, Saguinus sp., Saimiri sp., and Sapajus sp.) primates are important models for biomedical research and studies on reproductive physiology and biotechnology. Consequently, studies about gynecological and obstetric ultrasonography are crucial. B-mode ultrasonography is a non-invasive imaging technique that provides real-time bidimensional or three-dimensional/four-dimensional B-mode images. In association with Doppler ultrasonography, B-mode ultrasonography can also be used to monitor the mammalian blood flow to the reproductive tract during important events such as ovulation and gestation. Thus, gynecological and obstetric ultrasonography is essential for establishing the female reproductive anatomical and physiological ovarian and uterine health status, gestational diagnosis, and fetal growth monitoring. For instance, the paper presents and discusses the state-of-the-art gynecological and obstetric ultrasonography in the Neotropical primates, species that are models for biomedical research, and some recent studies on species targets for conservation strategies for wild animal populations.

Kinetic vitrification: concepts and perspectives in animal sperm cryopreservation

Sperm cryopreservation is an important tool for genetic diversity management programs and the conservation of endangered breeds and species. The most widely used method of sperm conservation is slow freezing, however, during the process, sperm cells suffer from cryoinjury, which reduces their viability and fertility rates. One of the alternatives to slow freezing is vitrification, that consist on rapid freezing, in which viable cells undergo glass-like solidification. This technology requires large concentrations of permeable cryoprotectants (P- CPA's) which increase the viscosity of the medium to prevent intracellular ice formation during cooling and warming, obtaining successful results in vitrification of oocytes and embryos. Unfortunately, this technology failed when applied to vitrification of sperm due to its higher sensitivity to increasing concentrations of P-CPAs. Alternatively, a technique termed 'kinetic sperm vitrification' has been used and consists in a technique of permeant cryoprotectant-free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen. Some of the advantages of kinetic vitrification are the speed of execution and no rate-controlled equipment required. This technique has been used successfully and with better results for motility in human (50-70% motility recovery), dog (42%), fish (82%) and donkey (21.7%). However, more studies are required to improve sperm viability after devitrification, especially when it comes to motility recovery. The objective of this review is to present the principles of kinetic vitrification, the main findings in the literature, and the perspectives for the utilization of this technique as a cryopreservation method.(AU)

Morphological and ultrastructural changes in seminal coagulum of the squirrel monkey (Saimiri collinsi Osgood, 1916) before and after liquefaction.

Studies with squirrel monkey semen are of special interest due to the large amount of coagulation that is a component of the semen, which is a problem that has to be overcome when the objective is harvesting of gametes. In the present study, there was characterization of the seminal coagulum of captive S. collinsi. Four samples of ejaculates were collected using electroejaculation procedures from four animals. The aim in conducting this study was to evaluate seminal coagulum of S. collinsi using histological and scanning electron microscopy (SEM) procedures before and after semen liquefaction in an ACP-118® extender. Seminal coagulum of S. collinsi was composed of a superficial plate (external), which coats the spongy seminal plasma matrix of S. collinsi. Additionally, there were sperm in the external and internal components of the coagulum with these gametes being isolated or grouped and with there being a heterogeneous distribution of gametes. The supplementation of semen with ACP-118® resulted in a partial dissolution of the seminal plate and spongy matrix portions of the seminal coagulum within the first hour of incubation.

Estudo preliminar da morfometria da cabeça espermática do macaco-de-cheiro de vida livre / Preliminary study of the spermatic head morphometry of free living Ecuadorian Squirrel Monkey

The objective of this study was to describe and compare the head morphometry of normal and pathological sperm from Saimiri macrodon. In the morphological analysis, 39% of the sperm had normal morphology, and 61% had major or minor defects, with pathology in the tails being the most frequent with 47% (38% curled tail, 7% folded tail and 2% strongly folded tail). Among the evaluated head morphometry parameters, area (A), width (L) and ellipticity (E) showed statistical difference (p>0.05) between normal and pathological sperm. The average head area and width was lower in normal sperm (p=0,01 e p=0,04, respectively), and the mean ellipticity was higher (p=0,038), when compared to pathological sperm. This definition of the sperm morphometric parameters of S. macrodon is important for the samples selection destined to reproduction biotechnologies and for the clarification of taxonomic and evolutionary issues in the genus Saimiri.

Biotecnologias da reprodução sob a perspectiva dos machos de primatas neotropicais: contribuições para a conservação de espécies ameaçadas de extinção / Reproduction biotechnologies for neotropical primate males and contributions to the conservation of endangered species

As biotecnologias da reprodução animal são uma alternativa para a conservação ex situ de espécies de primatas neotropicais ameaçados de extinção, e também podem ser utilizadas como uma ferramenta para o estudo da sua biologia reprodutiva. O entendimento dos mecanismos pré/pós copulatórios e pós-fertilização em primatas neotropicais podem auxiliar no sucesso do estabelecimento de protocolos de criopreservação seminal, visto que são necessários estudos de acordo com as especificidades fisiológicas de cada espécie. No intuito de realizar uma reflexão a acerca do papel do macho no tocante as biotecnologias da reprodução, esta revisão busca destacar os avanços e perspectivas a esse respeito, bem como as principais particularidades no tocante a manipulação do sêmen desses animais, visando uma reflexão acerca de melhorias de protocolos já estabelecidos, e fornecer uma referência para estudos futuros em primatas neotropicais.

The biotechnologies of animal reproduction are an alternative for the ex situ conservation of neotropical primates in some degree threatened with extinction, and could also be used as a tool for the study of reproductive biology. Understanding the pre/post copulatory and post-fertilization mechanisms in neotropical primates can help in the successful establishment of seminal cryopreservation protocols, since studies are necessary according to the physiological specificities of each species. In order to reflect on the role of the male in the reproductive biotechnologies, this review aim to highlight the advances and perspectives, as well as the main particularities with the manipulation of the semen in this animals, aiming at a reflection of improvements to already established protocols, and provide a reference for future studies in neotropical primates.

Análise morfométrica da cabeça espermática em Saimiri cassiquiarensis / Morphometric analysis of the sperm head in Saimiri cassiquiarensis

The objective was to evaluate the sperm morphometry between normal and pathological cells in Saimiri cassiquiarensis aiming at the future use of data in CASA (Computer Assisted Sperm Analysis). Semen was collected from a male (S. cassiquiarensis) by electroejaculation. After collection, a smear in the proportion of 1:1 (semen and eosin-nigrosin stain) was performed, and 100 sperm were measured and morphologically classified as normal and pathological defects (major and minor). Of the total sperm analyzed, 48% were classified as normal and 52% pathological. Of the pathologies, the most frequent was a folded tail (40%), followed by a strongly folded tail (5%), curled tail (2%) and isolated head (5%). The morphometric data showed no statistical difference (p<0.05) between normal and pathological defects. This is the first description for the sperm morphometry in Saimiri cassiquiarensis, therefore, from this database, future assessments at CASA can be conducted for this species.

Biotecnologias da reprodução sob a perspectiva dos machos de primatas neotropicais: contribuições para a conservação de espécies ameaçadas de extinção / Reproduction biotechnologies for neotropical primate males and contributions to the conservation of endangered species

As biotecnologias da reprodução animal são uma alternativa para a conservação ex situ de espécies de primatas neotropicais ameaçados de extinção, e também podem ser utilizadas como uma ferramenta para o estudo da sua biologia reprodutiva. O entendimento dos mecanismos pré/pós copulatórios e pós-fertilização em primatas neotropicais podem auxiliar no sucesso do estabelecimento de protocolos de criopreservação seminal, visto que são necessários estudos de acordo com as especificidades fisiológicas de cada espécie. No intuito de realizar uma reflexão a acerca do papel do macho no tocante as biotecnologias da reprodução, esta revisão busca destacar os avanços e perspectivas a esse respeito, bem como as principais particularidades no tocante a manipulação do sêmen desses animais, visando uma reflexão acerca de melhorias de protocolos já estabelecidos, e fornecer uma referência para estudos futuros em primatas neotropicais.(AU)

The biotechnologies of animal reproduction are an alternative for the ex situ conservation of neotropical primates in some degree threatened with extinction, and could also be used as a tool for the study of reproductive biology. Understanding the pre/post copulatory and post-fertilization mechanisms in neotropical primates can help in the successful establishment of seminal cryopreservation protocols, since studies are necessary according to the physiological specificities of each species. In order to reflect on the role of the male in the reproductive biotechnologies, this review aim to highlight the advances and perspectives, as well as the main particularities with the manipulation of the semen in this animals, aiming at a reflection of improvements to already established protocols, and provide a reference for future studies in neotropical primates.(AU)

Análise morfométrica da cabeça espermática em Saimiri cassiquiarensis / Morphometric analysis of the sperm head in Saimiri cassiquiarensis

The objective was to evaluate the sperm morphometry between normal and pathological cells in Saimiri cassiquiarensis aiming at the future use of data in CASA (Computer Assisted Sperm Analysis). Semen was collected from a male (S. cassiquiarensis) by electroejaculation. After collection, a smear in the proportion of 1:1 (semen and eosin-nigrosin stain) was performed, and 100 sperm were measured and morphologically classified as normal and pathological defects (major and minor). Of the total sperm analyzed, 48% were classified as normal and 52% pathological. Of the pathologies, the most frequent was a folded tail (40%), followed by a strongly folded tail (5%), curled tail (2%) and isolated head (5%). The morphometric data showed no statistical difference (p<0.05) between normal and pathological defects. This is the first description for the sperm morphometry in Saimiri cassiquiarensis, therefore, from this database, future assessments at CASA can be conducted for this species.(AU)

Contribuições da biotecnologia da reprodução sobre os desafios na manipulação do semên de primatas do gênero Saimiri / Reproductive biotechnology contributions on the challenges of semen manipulation in primates of the Saimiri genus

Algumas espécies de primatas do gênero Saimiri estão classificadas como vulneráveis ou quaseameaçadas. Diante desta realidade, é necessária a aplicação de alternativas para a conservação ex situ (emcativeiro) desses primatas, sobretudo o desenvolvimento de biotécnicas voltadas para a reprodução. Desta forma,o presente trabalho objetivou compilar informações, em linhas gerais, a respeito dos desafios, avanços eperspectivas em relação às pesquisas desenvolvidas no tocante à biotecnologia da reprodução em machos do gênero Saimiri.

Some species of Saimiri primates are classified as vulnerable or near threatened with extinction. Facingthis reality, it is necessary to apply alternatives for the ex situ conservation (in captivity) of these primates,especially the development of biotechniques of reproduction. In this way, the present study aimed to compileinformation, in general terms, regarding the challenges, advances and perspectives in relation to the researchesdeveloped regarding the biotechnology of reproduction in males of the genus Saimiri.

Contribuições da biotecnologia da reprodução sobre os desafios na manipulação do semên de primatas do gênero Saimiri / Reproductive biotechnology contributions on the challenges of semen manipulation in primates of the Saimiri genus

Algumas espécies de primatas do gênero Saimiri estão classificadas como vulneráveis ou quaseameaçadas. Diante desta realidade, é necessária a aplicação de alternativas para a conservação ex situ (emcativeiro) desses primatas, sobretudo o desenvolvimento de biotécnicas voltadas para a reprodução. Desta forma,o presente trabalho objetivou compilar informações, em linhas gerais, a respeito dos desafios, avanços eperspectivas em relação às pesquisas desenvolvidas no tocante à biotecnologia da reprodução em machos do gênero Saimiri.(AU)

Some species of Saimiri primates are classified as vulnerable or near threatened with extinction. Facingthis reality, it is necessary to apply alternatives for the ex situ conservation (in captivity) of these primates,especially the development of biotechniques of reproduction. In this way, the present study aimed to compileinformation, in general terms, regarding the challenges, advances and perspectives in relation to the researchesdeveloped regarding the biotechnology of reproduction in males of the genus Saimiri.(AU)