Abstract The objectives of the study were to (1) describe the kinematic parameters of spermatozoa (2) compare methods of evaluating sperm viability (3) validate assays of functionality and integrity of the sperm membrane and (4) evaluate possible changes between spermatozoa from the epididymis and the vas deferens of the greater rhea. Semen samples were recovered from 7 adult individuals. Sperm motility was characterized by adjusting the set-up for Computer-assisted semen analysis (CASA) to that new species. For sperm viability evaluation, smears of bromophenol blue and eosin-nigrosine dyes were used. Five solutions of different osmolarities were then tested for the hypoosmotic swelling test (HOST). The combination of fluorescent probes (propidium iodide - IP and Hoechst 33342) was also used to assess plasma membrane integrity. Data were presented as mean ± SEM. Rhea spermatozoa from the vas deferens had an overall motility of 14.6 ± 2.5%. The bromophenol blue staining technique revealed that 64.6 ± 5.2% sperm were viable, while that proportion was 72.1 ± 2.5% using eosin-nigrosine. An average of 77.6 ± 4.8% of spermatozoa reacted to the HOST with distilled water at 0 mOsm/l. Fluorescent probes indicated that 65.3 ± 2.6% of spermatozoa had intact membranes. Interestingly, no statistical differences were observed between the parameters analyzed in the epididymal spermatozoa and the vas deferens. These new assays set reference values that can now be used to further exploration of sperm handling conditions and freezing protocols in rheas.

Abstract Considering the relevance of establishing biodiversity conservation tools, the study aimed to investigate the TCM199 supplemented with different follicle-stimulating hormone (FSH) concentrations on survival and development of fresh and vitrified preantral follicles enclosed in red-rumped agouti ovarian tissues cultured in vitro. In the first experiment, six pairs of ovaries were fragmented and cultured for 6 days according to groups: 10 ng/mL pFSH (FSH10 group) and 50 ng/mL (FSH50 group). Non-cultured tissues were considered as a control. In the second experiment, vitrified/warmed fragments of four pairs of ovaries were cultured with the best concentration of FSH established (cryopreserved and cultured group). Non-cryopreserved (fresh control group) and cryopreserved but non-cultured (non-cultured group) tissues were used as controls. For both experiments, preantral follicles were evaluated for survival and development using morphological and viability analysis by trypan blue staining. After culturing fresh samples, FSH50 showed a higher percentage of morphologically normal follicles when compared to FSH10 (P < 0.05). This same response was observed for primordial follicles. Regardless of the concentrations of FSH used during in vitro culture, no difference was observed regarding the percentage of viable follicles and diameters (P > 0.05). Thus, the FSH50 group was used for second experiment, in which 76.2 ± 7.2% normal preantral follicles previously vitrified was found after 6-day culture, also presenting the highest values (P < 0.05) for morphology of primordial follicles (95.2 ± 4.7%). Nevertheless, in vitro culture did not affect the viability and diameter of preantral follicles of cryopreserved tissues (P > 0.05). In conclusion, TCM199 supplemented with 50 ng/mL FSH was efficient in maintaining the in vitro survival of fresh and vitrified red-rumped agouti preantral follicles. This was the first study related to the in vitro culture of ovarian preantral follicles in this species, aiming to contribute to its conservation.

Abstract Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.

Abstract Biological Resource Banks (BRB) or Genetic Resource Banks (GRB) are critical tools for the conservation of animal biodiversity. According to the International Union for Conservation of Nature, more than 38,500 species are threatened with extinction, out of a total of 138,300 surveyed species. These banks are repositories of biological samples and data recovered and preserved for the long term by zoos, universities, research centers and other conservation organizations. In recent years, BRB have increasingly included ovarian and testicular tissues as additional options to rescue and propagate wild species, especially those at risk of extinction. After in vitro culture or grafting, gonadal tissues are potential sources of matured gametes that can be used for Assisted Reproduction Technologies while informing about gametogenesis or mechanisms involved in infertility. It therefore is crucial to properly recover, cryopreserve, and culture these tissues using species-specific protocols. Developing BRBs is currently one of the strategies to preserve species from the Caatinga biome - an exclusively Brazilian biome with a rich wild fauna that suffers from anthropogenic activities. Among wild species from this biome, studies have been primarily conducted in collared peccaries, agoutis, cavies, and armadillos to preserve their ovarian and testicular tissues. Additionally, domestic species such as the domestic cat and donkeys have been proposed as models for wild species that are phylogenetically close. This review addresses the main technical aspects involved in obtaining BRB derived from gonadal tissues in some wild species of the Caatinga biome. It reports recent advances and perspectives to use these biological materials for wildlife conservation.

Abstract Myomorphic and hystricomorphic rodents are vital for maintaining various ecosystems around the planet. This review enables a better understanding of how these rodents respond to environmental factors and adapt to climate adversities. Innumerable factors, such as photoperiod, rainfall, and temperature, can impair or contribute to the quality of rodent reproductive parameters. Prolonged animal exposure to high ambient temperatures alters thermoregulation mechanisms and causes testicular and ovarian tissue degeneration and hormonal deregulation. Photoperiod influences the biological circannual rhythm and reproductive cycles of rodents because it strongly regulates melatonin secretion by the pineal gland, which modulates gonadotropic hormone secretion. Rainfall quantity directly regulates the abundance of fruits in an ecosystem, which modulates the reproductive seasonality of species which are most dependent on a seasonal fruit-based diet. Species with a more diversified fruit diet have smaller reproductive seasonality. As such, habitats are chosen by animals for various reasons, including the availability of food, sexual partners, intra-and inter-specific competition, and predation. This knowledge allows us to monitor and establish management plans to aid in conservation strategies for wild rodent species.

Abstract Studies on semen and sperm cells are critical to develop assisted reproductive technologies for the conservation of the collared peccary. The objective of the study was to compare the effect of different antibiotics on the bacterial load and sperm quality during short-term storage of peccary semen. Fresh semen samples from 10 males were extended in Tris-egg yolk or Tris-Aloe vera supplemented with streptomycin-penicillin (SP; 1 mg/mL - 1000 IU/mL or 2 mg/mL - 2000 IU/mL) or gentamicin (30 µg/mL or 70 µg/mL) before storage at 5°C. Bacterial load and sperm motility, membrane integrity and function, mitochondrial activity, and morphology, were evaluated at different time points for 36 h. The SP and gentamicin treatments concentration inhibited (p < 0.05) bacterial growth for 36 h regardless of the extender. Compared to the other treatments, Tris-egg yolk plus 70 µg/mL gentamicin maintained the sperm parameters for longer, including total motility (41.9 ± 6.1%) at 24 h, and membrane integrity (58.3 ± 2.1%) at 36 h. In contrast, the highest SP concentration in both extenders impaired sperm membrane integrity at 36 h (p < 0.05). For the liquid storage of collared peccary semen, it therefore is recommended to use Tris extender supplemented with egg yolk and gentamicin (70 µg/mL).

Abstract The use of antibacterial substances as additives in extenders for ensuring the sanitary quality of the semen employed in reproductive biotechniques and preserving it from bacterial deterioration has been reported since the mid-twentieth century. However, the deleterious effects of these drugs on the sperm quality as well as their effectiveness in controlling bacterial growth in the preserved semen have been questioned. The aim of this review was to report the antimicrobials primarily used in the extenders added to the semen of mammals, and to present alternatives to their use. Among the various mammalian species, there is a large variation regarding the antimicrobial types added to semen extenders as cephalosporins (ceftiofur, cefdinir, eg) and quinolones (ofloxacin, ciprofloxacin), alone or in combination with large action spectra substances as penicillin-streptomycin and gentamicin-tylosin-lincomycin-spectinomycin. To combat problems related to bacterial resistance to these drugs, the emergence of alternatives is increasingly evident. Among these alternatives, use of physical methods as centrifugation and filtration, as well as the use of antimicrobial peptides and other substances from different origins have been highlighted for presenting antimicrobial potential.

Abstract This study evaluated the effect of the extract of Aloe vera at concentrations of 10% and 20% on the cryopreservation of sperm from the epididymis of domestic cats. Epididymal spermatozoa were recovered using the flotation technique and used in the treatments: control (TRIS-egg yolk at 20%), T10% (TRIS plus 10% of A. vera extract), and T20% (TRIS plus 20% of A. vera extract). The spermatozoa were subjected to 4ºC for 60 minutes, followed by 20 minutes in nitrogen vapors, and stored in a cryogenic cylinder. The samples were thawed at 37°C for 30 seconds. The sperm motility decreased (P<0.05) after thawing in the three treatments. Only the spermatozoa in the control treatment maintained post-thawing vigor. The viability of spermatozoa decreased in the treatments with A. vera (P<0.05). According to the hypoosmotic test, all treatments maintained the sperm membrane functionality (P>0.05) during freezing; however, after thawing, it decreased (P<0.05) in the T10% and T20% treatments. The morphology and chromatin condensation of spermatozoa did not differ, regardless of the treatments and time of evaluation (P>0.05). The effect of the crude A. vera extract was not satisfactory on the cryopreservation of epididymal spermatozoa of domestic cats after thawing; although the motility of spermatozoa was similar to that found with the use of egg yolk, and it presented maintenance of the chromatin integrity. However, it is necessary to understand the action of the substances present in A. vera with the feline spermatozoa, well as the standardization and adjustment of physicochemical characteristics aiming at the future application of the vegetal extract.

Resumo: Este estudo foi realizado para determinar o efeito de hidroxitolueno butilado (BHT) sobre a qualidade do sêmen canino congelado e descongelado, utilizando o diluidor à base de água de coco em pó (ACP-106c). Para tanto, foram realizadas quinze coletas de sêmen provenientes de cinco cães. O sêmen obtido foi diluído em ACP-106c acrescido de glicerol e gema de ovo. As amostras foram então transferidas para tubos contendo diferentes concentrações de BHT (0; 0,5; 1,0 e 2,0 mM). Em seguida, as amostras foram envasadas, congeladas e armazenadas em nitrogênio líquido. O sêmen coletado foi avaliado in natura quanto aos seguintes parâmetros: coloração, volume da fração espermática, motilidade total, vigor, concentração, morfologia e funcionalidade de membrana espermática. Após uma semana, as amostras foram descongeladas e avaliadas por meio de análise computadorizada, como também foram realizadas análises da funcionalidade de membrana e da morfologia espermática. A motilidade progressiva no grupo BHT 2,0 mM foi significativamente superior (P < 0,05) do que a do grupo BHT 0 mM (27,6 ± 11,7% vs. 19,0 ± 9,5%, respectivamente). Em todos os demais parâmetros avaliados, não houve diferença entre os grupos testados. Portanto, conclui-se que a adição do BHT ao diluidor ACP-106c não afetou a qualidade do sêmen canino pós-descongelação.

Abstract: This study was conducted to determine the effect of butylated hydroxytoluene (BHT) on the quality of canine sperm frozen and thawed using the powdered coconut water based (ACP-106c) extender. Therefore, fifteen ejaculates were collected from five dogs. Semen obtained was diluted in ACP-106c added of glycerol and egg yolk. The samples were then transferred to tubes containing different concentrations of BHT (0, 0.5, 1.0 and 2.0 mM). After that, the samples were filled into straws, frozen and stored in liquid nitrogen. Fresh semen was evaluated for the following parameters: color, sperm fraction volume, total motility, vigor, concentration, morphology, and HOST test. After one week, the samples were thawed and evaluated by computer analysis, as well as for membrane functionality and sperm morphology. Progressive motility in the 2.0 mM BHT group was significantly higher (P <0.05) than that of the 0 mM BHT group (27.6 ± 11.7% vs. 19.0 ± 9.5%, respectively). Regarding all other parameters evaluated, there was no difference between the groups tested. Therefore, the addition of BHT to the ACP-106c extender did not affect the quality of canine semen after thawing.

Foram avaliados os efeitos da centrifugação associada ao uso de dois diluentes na manutenção da viabilidade espermática em tatus-peba (Euphractus sexcinctus) ao longo do teste de termorresistência (TTR). Amostras de sêmen (n=12), oriundas de 04 machos adultos coletados por eletroejaculação, foram divididas em quatro alíquotas, sendo duas imediatamente diluídas em Tris ou água de coco em pó (ACP-119(r)), e as outras duas centrifugadas (800g10min-1) previamente à diluição. As amostras foram incubadas a 34°C por 3h, e os parâmetros seminais avaliados em intervalos de 1h. Em termos gerais, verificou-se uma redução da viscosidade espermática imediata à diluição em ambos os diluentes, independente do uso da centrifugação. Aos 60 minutos, verificou-se uma redução dos parâmetros avaliados (P<0,05), embora o Tris tenha promovido uma melhor preservação deles (P<0,05), quando comparado ao ACP-119(r) até os 120 minutos de avaliação. Após este período, os dois diluentes se equipararam (P>0,05). Ainda, verificou-se um efeito deletério da centrifugação sobre a qualidade do sêmen de tatus-peba durante todo o teste de termorresistência. Nas condições do presente estudo, conclui-se que o diluente Tris mostrou-se superior ao ACP-119(r) para a manutenção da viabilidade do sêmen de tatus-peba, sendo desnecessária a realização de centrifugação prévia à diluição.

The effects of the centrifugation associated to the use of two extenders on the viability of six-banded armadillo's (Euphractus sexcinctus) sperm were evaluated during a thermo resistance test (TRT). Semen samples (n=12) derived from 04 stud males collected by electroejaculation were divided in four aliquots; two of that were immediately diluted in Tris or powdered coconut water (ACP-119(r)); the two others were centrifuged (800g10min-1) prior to the dilution. Samples were incubated at 34ºC per 3h, and the semen parameters were evaluated at each hour. In general, dilution promoted a reduction in semen viscosity in the use of both diluents using centrifugation or not. At 60min, a decrease was verified for all semen parameters (P<0.05), however they were better preserved in the use of Tris when compared to ACP-119(r) (P<0.05) up to 120min. After that, both diluents equated. In addition, centrifugation procedure presented a deleterious effect on the armadillo's semen quality during all the thermoresistance test. In the present conditions, conclude that Tris extender is more efficient than ACP-119(r) for the preservation of six-banded armadillo semen viability, and the previous centrifugation is unnecessary.

Em ejaculados provenientes de 28 catetos, verificou-se a existência de relações entre a concentração espermática determinada por meio da câmara de Neubauer e a tramitância observada por espectrofotometria, utilizando comprimentos de onda variando de 530 a 590nm. Os ejaculados apresentaram uma concentração média de 283,9±30,8x106 espermatozoides mL-1, com variação de 30 a 640x106 espermatozoides mL-1. Os valores para tramitância variaram entre 36,9 a 96,3, nos diferentes comprimentos de onda. Não foram detectadas relações significativas entre os dois métodos (P>0,05). Dessa forma, não se recomenda a espectrofotometria para os procedimentos de rotina na determinação da concentração espermática em catetos.

In ejaculates derived from 28 collared peccaries, we verified the existence of relationships between sperm concentration determined by the Neubauer counting chamber and the tramitance verified by spectrophotometer, under wavelengths varying from 530 to 590nm. Ejaculates presented a concentration of 283.9±30.8x106sperm m-1, varying from 30 to 640x106sperm mL-1. Values for tramitance varied from 36.9 to 96.3, under different wavelengths. No significant relationship was verified between two methods (P>0.05). Thus, the spectrophotometer is not recommended for routine procedures of sperm concentration measurement in collared peccaries.

Avaliou-se a correlação entre a avaliação da integridade funcional de membrana espermática por meio do teste hiposmótico, utilizando-se soluções com diferentes osmolaridades, e as avaliações convencionais do sêmen fresco de caprinos. Foram utilizados 24 ejaculados de três caprinos machos, obtidos por vagina artificial, os quais foram imediatamente submetidos à avaliação clássica. Em seguida, as amostras foram divididas em cinco alíquotas e submetidas ao teste hiposmótico, utilizando-se água destilada (0 mOsm/L) e soluções de citrato de sódio e frutose de diferentes osmolaridades (50; 100; 150 e 200 mOsm/L). A solução de 100 mOsm/L obteve a maior média de espermatozoides reagidos (34,8%) ao teste hiposmótico (P < 0,05); por outro lado, a água destilada foi a que apresentou os menores valores (20,8%). Não foram detectadas correlações significativas entre os espermatozoides reagidos ao teste hiposmótico e as demais características seminais (P > 0,05). Apesar disso, recomenda-se que o teste hiposmótico seja realizado utilizando-se uma solução a base de citrato de sódio e frutose apresentando 100 mOsm/L para a avaliação da integridade funcional da membrana espermática na espécie caprina.

We evaluated the correlation between the assessment of the functional integrity of the sperm membrane by hypoosmotic swelling tes,t using solutions with different osmolarities, and the conventional assessments of fresh semen in goats. A total of 24 ejaculates from three goats was obtained by artificial vagina and immediately submitted to the classical evaluation. Samples were divided into five aliquots and subjected to hypoosmotic test using distilled water (0 mOsm/L), and sodium citrate and fructose solutions at different osmolarities (50; 100; 150 and 200 mOsm/L). The 100 mOsm/L solution showed the highest percentage of reacted sperm (34.8%), but distilled water was the one with the lowest values (20.8%). No significant correlations were detected between the reacted sperm verified by the hypoosmotic swelling test and other semen characteristics (P> 0.05). Nevertheless, we recommend the carry out of the hypoosmotic test by using a 100 mOsm/L sodium citrate and fructose solution to assess the functional integrity of the sperm membrane in caprine species.

The objective of this study was to analyze interactions between different straw sizes and thawing rates on the post-thaw goat semen parameters. Twenty-one ejaculates (seven per animal) were collected from three stud bucks by using an artificial vagina. After evaluation, the semen was extended in Tris-egg yolk-glycerol and packed in 0.25 and 0.50 mL straws, followed by storage in liquid nitrogen. Thawing was performed using two different rates: 37 ºC/1 min and 55 ºC/7 s. The interaction between the 0.5-mL straw and the thawing rate of 55 ºC/7 s promoted higher progressive motility. When the effect of straws alone was analyzed, it was verified that the use of the 0.50 mL straw promoted better conservation than the 0.25 mL one for progressive motility and acrosomal integrity, after the frozen-thawing procedures. Optimal results for progressive motility were achieved when goat semen was frozen in 0.5 mL straws and thawed in water at 55 ºC/7 s.

BACKGROUND: Elderly people present alterations in body composition and physical fitness, compromising their quality of life. Chronic diseases, including HIV/AIDS, worsen this situation. Resistance exercises are prescribed to improve fitness and promote healthier and independent aging. Recovery of strength and physical fitness is the goal of exercise in AIDS wasting syndrome. OBJECTIVE: This study describes a case series of HIV-positive elderly patients who participated in a progressive resistance training program and evaluates their body composition, muscular strength, physical fitness and the evolution of CD4+ and CD8+ cell counts. METHODS: Subjects were prospectively recruited for nine months. The training program consisted of three sets of 8-12 repetitions of leg press, seated row, lumbar extension and chest press, performed with free weight machines hts, twice/week for one year. Infectious disease physicians followed patients and reported all relevant clinical data. Body composition was assessed by anthropometric measures and dual-energy x-ray absorptiometry before and after the training program. RESULTS: Fourteen patients, aged 62-71 years old, of both genders, without regular physical activity who had an average of nine years of HIV/AIDS history were enrolled. The strengths of major muscle groups increased (74%-122%, p=0.003-0.021) with a corresponding improvement in sit-standing and walking 2.4 m tests (p=0.003). There were no changes in clinical conditions and body composition measures, but triceps and thigh skinfolds were significantly reduced (p=0.037). In addition, there were significant increases in the CD4+ counts (N=151 cells; p=0.008) and the CD4+/CD8+ ratio (0.63 to 0.81, p=0.009). CONCLUSION: Resistance training increased strength, improved physical fitness, reduced upper and lower limb skinfolds, and were associated with an improvement in the CD4+ and CD4+/CD8+ counts in HIV positive elderly patients without significant side effects.

This study reported a case of rhinosinusitis for Respiratory Syncytial Virus in Intensive Care Unit patient. The settings were Intensive Care Unit at Hospital das Clínicas, University of São Paulo Medical School, São Paulo, Brazil. One female HIV-infected patient with respiratory failure and circulatory shock due to splenic and renal abscesses, who developed rhinosinusitis caused by RSV and bacteria. Respiratory viruses can play a pathogenic role in airways infection allowing secondary bacterial overgrowth.