Criopreservação do sêmen de caprinos em diluidores alternativos e análise da viabilidade / Cryopreservation of goat semen in alternative extenders and viability analysis

Esse trabalho teve como objetivo avaliar os efeitos da refrigeração e congelação do sêmen caprino utilizando diluentes de origem vegetal. Para isso, foi utilizado 20 amostras de sêmen de cinco machos caprinos, em que foi avaliado imediatamente após a coleta, diluídos em ACP-101 e extrato liofilizado de Aloe vera e controle Tris. O sêmen foi refrigerado a 4 °C e congelado a -196 °C. Ao término da refrigeração e após descongelação as amostras foram avaliadas quanto aos parâmetros cinéticos de motilidade, velocidades e viabilidade. Os resultados das avaliações, demonstraram que o diluente ACP apresentou resultados superiores em comparação aos tratamentos com Aloe vera. Portanto, a utilização do ACP-101 associado aos crioprotetores proporciona um excelente meio para criopreservação do sêmen de caprinos. Novos estudos são necessários para verificar em que concentração o aloe vera pode ser utilizado na criopreservação do semen exercendo outros beneficios

Influence of Coconut Powder Water-Based Conservation Medium (APC-102c) for Maintaining Mitochondrial Activity of Cryopreserved Ram Sperm

Background:  Semen extenders are required to protect and preserve semen, and the development of suitable extenders is key for artificial insemination. Although the use of Tris-based diluent is widespread, new diluents such as powdered coconut water have been developed for better sperm protection. One way to evaluate the effectiveness of diluents is through microscopic analyses that evaluate sperm motility, vigor, and concentration. However, these analyses are limited, and may not provide accurate results. New evaluation techniques have been studied, and one of the tests that can be used to add reliability to these analyses is mitochondrial activity evaluation, which can sum all the parameters, and provide a more accurate evaluation. Thus, the present study aimed to evaluate the efficacy of ACP-102c in cryopreserved ram semen.Materials, Methods & Results: Five semen samples were collected from two ram breeders using artificial vagina (n = 10). Each ejaculate was divided into the following two treatments: T1 - ACP-102c + 20% egg yolk + 7% glycerol and T2 - TRIS + 20% egg yolk + 7% glycerol. Extended semen samples were then packed in 0.5 mL plastic straws, subjected through the refrigeration curve up to 4°C (0.35° C/min), and equilibrated for 2 h at 4°C. Subsequently, the straws were placed at 4 cm above liquid nitrogen level (-60°C) for 15 min, immersed, and then finally stored in the liquid nitrogen at -196°C. Both fresh and thawed samples were evaluated for total and progressive sperm motility using conventional microscopy (40x), and the same evaluator on each occasion. For plasma membrane integrity (IMP), the smear staining technique with the Eosin-Nigrosin staining was used; 200 sperms were counted and classified as whole (unstained) and unhealthy (stained). Mitochondrial activity was evaluated using a cytochemical technique based on the oxidation of 3,3'-diaminobenzidine (DAB); 200 sperms were counted, and classified into four classes (I, II, III, and IV) according to the degree of coloration of the intermediate part. Fresh semen showed no significant difference (P > 0.05) between treatments with respect to motility parameters; however, T2 showed significantly inferior results regarding plasma membrane integrity. After thawing, T2 was significantly higher in sperm motility parameters compared to T1. The mitochondrial activity and plasma membrane integrity parameters did not show any significant difference between the treatments.Discussion: The TRIS-based diluent showed higher motility values than ACP-102c; however, motility rates in ACP-102c diluent, although lower, are considered satisfactory for insemination, which requires semen with minimal progressive motility of 30%. Notably, the cryopreservation protocol used in this study is the standard for TRIS-based diluent, and it is known that the optimal rate of refrigeration and cryopreservation may differ according to the composition of the storage medium; therefore, we may assume that the protocol used is not yet appropriate for the ACP-102c diluent, and further studies are required. IMP is an essential attribute for fertilization, and cryopreservation can affect the plasma membrane as observed in this study. Cryopreserved semen reduced the percentage of class I mitochondrial reaction sperms in both treatments, demonstrating that cryopreservation affects the mitochondrial activity of the intermediate portion of the sperm; however, there was no difference between treatments in thawed semen. Thus, we concluded that the ACP-102c conservation medium maintains seminal quality after thawing, and it can be used in artificial insemination processes

Ram and Goat Semen Immunosexed and Diluted in Powdered Coconut Water-Based Preservation Medium (ACP101/102c).

Background: Sperm sexing aims to separate sperm populations in carriers of the “X” or “Y” chromosome. Currently, flow cytometry is a technique that allows greater accuracy; however, it causes structural changes in sperm, reduces viability, and has a high cost. As a result, other methods have been researched, including immunosexing, which uses monoclonal antibodies to detect sex-specific surface antigens. Thus, the objective of this study was to evaluate the immunosexing technique using a monoclonal antibody against sex-specific protein (HY) in the conservation of ram and goat semen in ACP101/102c.Materials, Methods & Results: Ejaculates from 5 rams and 5 goats were collected with the aid of an artificial vagina; they were evaluated and submitted to the immunosexing protocol, according to the manufacturer's recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with “Y” chromosomes (HY; HY Biotechnology, Rio de Janeiro, RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP ram (ACP101/102c + 20% egg yolk + 7% glycerol) and ACP goat (ACP101/102c + 2.5% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated at 4°C, stabilized for 30 min, frozen in liquid nitrogen vapor (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were evaluated in natura (T1), after immunosexing (T2) and after thawing (T3) for sperm motility subjectively using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and morphologically defect spermatozoa were determined. In the evaluation of ram semen regarding sperm motility and IMP, no statistically significant differences were observed between treatments after sexing in the evaluation of absolute data (P > 0.05), with the difference being observed only between T1 and T2, and T3 (P 0.05). Regarding the evaluation of goat semen samples, the motility parameters were consistent with the technique submitted; however, the IMP data did not appear as expected, requiring further evaluation for a better assessment of the technique for this species.Discussion: The data obtained from ram semen submitted to the immunosexing protocol, regarding the absolute evaluation of motility and IMP, demonstrated that the non-sexed semen (T1) was superior to the sexed treatments (T2 and T3); however, it is noteworthy that freezing started with approximately 50% of the cells, since the immunosexing technique results in a loss of viability of approximately 50% of the sperm, which corresponds to the ratio of sperm carrying the X chromosome. In addition, when the data in this study were transformed into relative values, no statistical differences were observed, indicating that the immunosexing protocol, as well as the freezing protocol, did not significantly affect the quality of ram sperm cells. In relation to the immunosexing of goat semen, future studies should be conducted in vitro to define a more appropriate protocol for the species and, in addition, in vivo studies should be performed to prove the quality of the technique. It was concluded that the immunosexing process using a monoclonal antibody against sex-specific protein (HY) associated with the use of powdered coconut water diluent (ACP101/102c) in the cryopreservation of semen proved to be efficient in the in vitro evaluation of ovine species

Testicular Morphological and Ultrasonographic Characterization of Male Gray Brocket Deers (Mazama gouazoubira) in Different Reproductive Status

Background: Gray brocket deer (Mazama gouazoubira) populations have been declining due to human intervention. Yet, only a few studies have assessed ultrasonographic testicular characteristics in cervids. Considering the relevance of monitoring testicular size, blood flow, and parenchyma, the present study aims to establish baseline information on scrotal circumference, testicular volume, and spectral Doppler parameters, to describe differences among adult male gray brocket deer in different reproductive status, and to correlate ultrasound parameters with testes size measurements.Materials, Methods & Results: Six adult male gray brocket deers were used in the study. Scrotal circumference and testicular volume were measured. B mode ultrasound images of testes (longitudinal and cross-sectional views) and epididymes were subjected to computer-assisted analysis, obtaining the numerical pixel values (NPV) and pixel standard deviation (PSD). Using spectral Doppler, supratesticular artery blood flow velocities (peak systolic velocity - PSV, end diastolic velocity - EDV, time-average maximum velocity - TAMAX, resistivity - RI and pulsatility indices - PI) were obtained. Semen was analyzed through total motility, vigor, and concentration tests. Three animals were normospermic (F+ group) and three were oligo/azoospermic (F- group). Groups were compared using were compared using a one-way ANOVA or Kruskal-Wallis followed by Student-Newman-Keuls (SNK) test. Ultrasound parameters were correlated to testes size parameters using Pearson’s correlation for parametric variables and Spearman’s correlation for non-parametric variables. F+ group presented significantly higher scrotal circumference (14.57 ± 1.19 cm), testicular volume (26.18 ± 4.94 cm3), and testes cross-sectional NPV (69.88 ± 24.00) and PSD (10.78 ± 3.42) than group F- (NPV: 28.26 ± 13.75, PSD: 6.70 ± 1.84). No significant differences were observed between the groups regarding the spectral Doppler ultrasound parameters. Significant correlations were observed between scrotal circumference and longitudinal (r = 0.76) and cross-sectional testes NPV (r = 0.89), and testicular volume was correlated with longitudinal (r = 0.78) and cross-sectional testes NPV (r = 0.91) and with cross-sectional testes PSD (ρ = 0.82).Discussion: Increased testicular echogenicity (higher NPV) has been positively associated with improved testicular growth, cell population expansion, inner and outer seminiferous tubules diameter, spermatids percentages and testis weight. In addition, more heterogenous testes (higher PSD) were associated with higher sperm output. It was suggested that the animals in group F- had compromised testicular development and spermatogenesis. The correlation observed between testes NPV and scrotal circumference was proposed to be associated with seminiferous tubules impairment. The F- group showed lower testicular volume, NPVs and PSDs in cross-sectional testicular images, suggesting higher protein levels and lower lipid contents were present in their parenchyma, influencing in testicular echogenicity and echotexture. No differences in spectral Doppler parameters were observed between the two groups. Also observed in stallions. However, PSV, EDV, TAMAX could be potential infertility indicators in other mammalians. These different results may be due to different locations of the evaluated vessel, species and techniques, age, ambient temperature, pathological conditions, and anaesthesia. Thus, it is suggested that scrotal circumference, testicular volume, and testes NPV are good indicators of male reproductive health in gray brocket deer and may help with better male selection in the species

Economic Aspects and Evaluation of the Viability Period of Immunosexed and Diluted Ram Sperm in Powdered Coconut Water Conservation Extender (ACP-102c)

Background: Sperm sexing is increasing in use because pre-determining the sex of the calf allows greater profitability and promotes significant gains in the productive systems that utilize the technique. Deployment of a low-cost and practical preservation methodology may further favor the cost-benefit ratio. Flow cytometry, the most commonly used sexing technique, has high costs and is very restricted. As an alternative, immunosexing has been studied, which uses sex-specific monoclonal antibodies. Thus, the objective of this study was to evaluate the immunosexing technique in conjunction with cryopreservation in ACP-102c and examine its economic aspects with regard to ram semen.Materials, Methods & Results: Ejaculates from two ram individuals were collected with the aid of an artificial vagina, evaluated, and submitted to the immunosexing protocol, according to the manufacturer's recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with “Y” chromosomes (HY; HY Biotechnology, Rio de Janeiro-RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP (ACP-102c + 20% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated to 4°C, stabilized for 30 min, frozen in liquid nitrogen vapors (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were thawed and evaluated for sperm kinetics both by using computerized semen analysis with SCA® software (Sperm Class Analyzer version 5.0) and subjectively comparing specimens from the two animals using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and normal spermatozoa were determined. A bibliographic survey and a market study of similar products and technologies were carried out to provide an economic viability metanalysis of the bioproduct (ACP-102c) and bioprocess (immunosex). The data were analyzed using R-project©, and comparisons made between animals and between thawing periods using T test. There were no statistically significant differences between animals and between periods (P > 0.05), except for the normal sperm parameter, in which animal A1 had the lowest percentage (P < 0.05). As for the cost-benefit ratio, flow cytometry as a technique is more laborious and expensive, while immunosexing associated with cryopreservation in ACP-102c diluent has proven more practical, with regards to both sperm sexing techniques and diluents for sperm conservation.Discussion: In general, the quality of cryopreserved sexed semen was lower than that of non-sexed semen; however, in this study, both in the comparison between animals and between evaluation periods, similar values of motility, viability, and sperm morphology were obtained for sexed and several non-sexed cryopreserved semen samples, demonstrating that immunosexing did not severely affect the sperm structure, and that the ACP-102c conservation medium was efficient at maintaining the plasma membrane of these sperm. In the evaluation of economic aspects, it was observed that immunosexing, associated with cryopreservation in ACP-102c diluent, proved to be the most practical technique, requiring only conventional equipment, and allowing a greater field of application, since the immunosexing semen can be used for primiparous and multiparous females. Thus, it was concluded that immunosexing associated with cryopreservation in an ACP-102c diluent was more cost-effective, more practical, and had significantly improved sperm quality results after sexing

Intoxicação natural por Leucaena spp em equinos no Município de Volta Redonda / Natural intoxication by Leucaena spp in horses in the Municipality of Volta Redonda

Leucena [Leucaena leucocephala (Lam.) R. de Wit] é uma leguminosa originária da América Central, amplamente utilizada na alimentação animal, por seu alto valor nutritivo e boa palatabilidade, porém, a presença do princípio tóxico, a mimosina, um aminoácido capaz de promover efeitos inibitórios e antagonistas de várias funções fisiológicas, restringe sua utilização. A intoxicação por leucena foi relatada em bovinos, equinos, caprinos, ovinos e coelhos; o principal sinal clínico é a perda de pelos em consequência a um quadro de hipotireoidismo. No Brasil foram descritos 4 relatos de intoxicação natural por essa planta em equinos, estudos que carecem de informações quanto às avaliações hematológicas. Este trabalho objetiva descrever a intoxicação por leucena em dois equinos fêmeas e avaliar os níveis séricos dos hormônios tireoidianos. Os resultados mostraram que a leucena foi capaz de alterar o hematócrito e promover anisocitose com predominância de hemácias microcíticas, bem como promoveu diminuição dos níveis de T4 livre e T4 total

ENTRE NARRATIVAS LITERÁRIAS E HISTÓRICAS: CONFLUÊNCIAS ENTRE O LIVRO DIDÁTICO DE HISTÓRIA E A LITERATURA

Este trabalho constitui-se de uma reflexão sobre umas das atividades didático-pedagógicas desenvolvidas pelo subprojeto A Formação da Consciência Histórica: articulações entre metodologia e prática no ensino de História, elaborado por um coletivo de professores do Curso de História da Faculdade de Filosofia Dom Aureliano Matos (FAFIDAM), campus da Universidade Estadual do Ceará (UECE) em Limoeiro do Norte na Escola de Ensino Médio Arsênio Ferreira Maia no ano de 2017, explorando as relações entre o livro didático de História e a Literatura reforçando a dimensão educativa da biblioteca escolar e as possibilidades de utilização de seu acervo