POTENCIAL BIOTECNOLÓGICO DA SEXAGEM ESPERMÁTICA NA PRODUÇÃO ANIMAL

The predetermination of progeny sex is a desire among breeders, since it allows the sexing of born animals according the purpose of the production system. With the discovery of the sex chromosomes X and Y, the sex pre-selection studies followed a scientific direction, in order to develop techniques for the separation of sperm populations with these chromosomes. For this purpose it was developed and improved the flow cytometer separation method, which is highly accurate, although presenting a high cost and causing sperm injuries. Therefore, alternative protocols have been sought to enable sperm sexing, without major structural and functional damage to gametes, with emphasis on Percoll® density gradients. Due to the biotechnological potential of sperm sexing, in order to maximize the animal production, it was objectified in this review expose the positive and negative points of the two main techniques used for this purpose.A predeterminação do sexo da progênie é um desejo entre os criadores, uma vez que permite o direcionamento do sexo dos animais nascidos, segundo a finalidade do sistema de produção. Com a descoberta dos cromossomos sexuais X e Y, estudos para a pré-seleção do sexo seguiram um direcionamento científico, a fim de desenvolver técnicas para a separação das populações de espermatozoides portadores destes cromossomos. Com esta finalidade foi desenvolvido e aprimorado o método de separação em citômetro de fluxo, que é de alta precisão, embora apresente custo elevado e cause injúrias aos espermatozoides. Por conseguinte, protocolos alternativos vêm sendo buscados para viabilizar a sexagem espermática, sem maiores prejuízos estruturais e funcionais aos gametas, no que se destacam os gradientes de densidade de Percoll®. Em decorrência do potencial biotecnológico da sexagem espermática, visando maximizar a produção animal, foi objetivado com esta revisão expor os pontos positivos e negativos das duas principais técnicas usadas para este fim

Sondas fluorescentes: um avanço na avaliação da integridade estrutural e funcional de espermatozoides

The semen quality is a determining factor for obtaining good results with reproductive biotechnologies, such as artificial insemination (AI). Thus, the evaluation of sperm parameters is essential to determine the fertility of gametes. Assessment techniques that best predict in vitro fertility of sperm have been relentlessly pursued with the purpose of ensuring better results after AI. However, until now, no evaluation methodology alone was efficient for this purpose. Then it is recommended to associate the use of these techniques to better predict the fertilizing ability of spermatozoa. In recent years a great number of new strategies were developed for semen evaluation, with the highlighted use of fluorescent probes. These techniques have limited use due to unfamiliarity with their employment. The aim of this paper is to review the advances on semen evaluation and prediction of their fertilising ability, with emphasis on those based on fluorescent probes.A qualidade do sêmen é um fator determinante para se obter bons resultados com a utilização de biotécnicas reprodutivas como a inseminação artificial (IA). Desta forma, torna-se essencial a avaliação dos parâmetros espermáticos que determinem a fertilidade destes gametas. Técnicas de avaliação que melhor predigam a fertilidade in vitro dos espermatozoides têm sido buscadas incessantemente, a fim de garantir melhores resultados após IA. Entretanto, até o momento, nenhuma metodologia de avaliação mostrouse efi ciente para este fim, quando utilizada isoladamente, sendo recomendado o emprego conjunto destas técnicas para a melhor predição da capacidade fertilizante dos espermatozoides. Nos últimos anos uma série de novas estratégias foram desenvolvidas para avaliação de sêmen, dentre as quais se destaca o emprego das sondas fluorescentes. Estas técnicas são utilizadas com limitações em função do desconhecimento de seu emprego. Esta revisão tem como objetivo abordar os avanços na avaliação do sêmen e a predição de sua capacidade fertilizante, com ênfase nas que se baseiam no uso de sondas fluorescentes

Congelação de sêmen caprino: : as duas faces da moeda

Although the niche market for goat breeding products is on the rise, this activity is still carried out in family and disorganized systems. Thus, technification of this sector is necessary, including the introduction of reproduction biotechnology, such as semen cryopreservation and artificial insemination (AI). Semen freezing enables the breeder to improve the genetic quality of their herds at a lower cost, increasing the male potential without limit of time or space, and facilitating breeding management when in association with AI. As a consequence, the productivity and profitability of this sector is elevated, which could favor the transition from a livestock subsistence system to an industrial one. Nevertheless, the use of frozen semen of goats still presents some limitations, since the extender and procedures used for this biotechnology subject the gametes to numerous structural and functional injuries, both lethal and sublethal. These factors are reflected in the fertility rates, which are reduced after AI with frozen goat semen. In this perspective, the objective of the current review work is to report the positive and negative aspects of semen freezing biotechnology, with emphasis on the goat species.O nicho consumidor de produtos da caprinocultura se encontra em plena ascensão. Apesar disso, tal atividade pecuária ainda é realizada em sistema familiar e de forma desorganizada. Deste modo, a tecnificação do setor é necessária, inclusive pela introdução de biotécnicas da reprodução, como a criopreservação de sêmen e a inseminação artificial (IA). O uso do sêmen congelado possibilita ao produtor melhorar a qualidade genética de seu rebanho a um menor custo, ampliar o potencial do reprodutor sem limite de tempo ou espaço e facilitar o manejo das criações, quando em associação a IA. Como consequência, são elevadas a produtividade e lucratividade do setor, o que inclusive favorece a transição de um sistema pecuário de subsistência para o industrial. Contudo, a congelação do sêmen caprino e o seu uso ainda apresenta certas limitações, visto que os diluidores e procedimentos empregados para esta biotécnica submetem os gametas à inúmeras injúrias estruturais e funcionais, letais e subletais. Tais fatos se refletem nas taxas de fertilidades, as quais se mostram reduzidas após a IA com sêmen congelado caprino. Diante dessa perspectiva, foi objetivado com este trabalho de revisão expor os pontos positivos e negativos da biotécnica da congelação de sêmen, com ênfase para a espécie caprina

Soybean lecithin-based extender as an alternative for goat sperm cryopreservation

AbstractThe aim of this study was to evaluate the effect of different concentrations of soybean lecithin (SL) in extenders for sperm goat cryopreservation. Sexually mature male Saanen goats (n=4) were used, and the ejaculates were obtained using an artificial vagina method. The semen samples were pooled and diluted in a skim milk-based extender (control group; CG) or Tris extender supplemented with SL at different concentrations (G1=0.04%, SL G2=0.08% SL and G3=0.16%) for a final concentration of 240×106spermatozoa/mL. The semen samples were packed in straws (0.25mL), frozen using an automated system and stored in liquid nitrogen (−196°C). After thawing (37°C/30s), the samples were evaluated for sperm quality parameters, including sperm motility, membrane integrity, acrosome integrity and mitochondrial activity. No significant difference was observed among the experimental and control groups for all of the parameters (P>0.05). However, even though the control group presented a significantly lower mitochondrial membrane potential compared to fresh semen (P<0.05), the same did not occur for the extender supplemented with soybean lecithin, that is, it did not differ from fresh sperm (P>0.05). The extender containing soybean lecithin at different concentrations preserved the sperm quality parameters in a manner similar to the conventional skim milk-based extender. Thus, it is concluded that an extender containing soybean lecithin as the lipoprotein source can be used for freezing goat semen

DILUENTE QUIMICAMENTE DEFINIDO E ANTIOXIDANTES POLIFENOIS NA CRIOPRESERVAÇÃO DE SÊMEN OVINO

The objective of this study was to elaborate chemically defined extender based on casein, added with polyphenolic antioxidants, for the freezing of semen from ram breeders. To evaluate Tris-casein (5% glycerol), plus polyphenols, four semen pools from three rams sires were frozen (0µM; 10µM resveratrol; 5µM quercetin; 25µM catechin; 25µM catechin + 10µM resveratrol; 25µM catechin + 5µM quercetin; 10 µM resveratrol + 5µM quercetin; 25µM catechin + 10µM resveratrol + 5µM quercetin). After thawing, the samples were evaluated for sperm kinetics, plasma and acrosomal membrane integrity and mitochondrial membrane potential. There was no significant difference (p≥0.05) between the control group and the treatments for any of the evaluated parameters. However, it can be seen that the Tris-casein extender was efficient in cryopreserving ram semen. It is concluded that the Tris-casein extender can be used for freezing ram semen.O objetivo deste trabalho foi elaborar diluidor quimicamente definido à base de caseína, acrescido de antioxidantes polifenólicos, para congelamento de sêmen de reprodutoras ovinas. Para avaliar Tris-caseína (5% glicerol), mais polifenóis, quatro pools de sêmen de três touros carneiros foram congelados (0µM; 10µM resveratrol; 5µM quercetina; 25µM catequina; 25µM catequina + 10µM resveratrol; 25µM catequina + 5µM quercetina; 10 µM resveratrol + 5µM de quercetina; 25µM de catequina + 10µM de resveratrol + 5µM de quercetina). Após o descongelamento, as amostras foram avaliadas quanto à cinética espermática, integridade da membrana plasmática e acrossômica e potencial da membrana mitocondrial. Não houve diferença significativa (p≥0,05) entre o grupo controle e os tratamentos para nenhum dos parâmetros avaliados. No entanto, pode-se observar que o diluidor Tris-caseína foi eficiente na criopreservação do sêmen ovino. Conclui-se que o diluidor Tris-caseína pode ser utilizado para congelamento de sêmen ovino

EFEITO DA ADIÇÃO DE NANOPARTÍCULAS DE ÓXIDO DE ZINCO AO DILUENTE DE CONGELAÇÃO DO SÊMEN OVINO

The aim of this study was to evaluate the effect of adding zinc oxide nanoparticles to the ram semen cryopreservation diluter. Semen pools (n=6), from three Santa Inês breeders, were diluted in Tris-yolk (5% glycerol), supplemented with zinc oxide nanoparticles (0, 25, 75 and 150μg/mL) at a concentration of 200x106 sperm/mL. The samples were frozen in an automated system and stored in liquid nitrogen (-196 °C). At the time of analysis, the semen samples were thawed (37 °C/30s) and evaluated for sperm kinetics, plasma and acrosomal membrane integrity and mitochondrial membrane potential. There was no significant difference (p&gt;0.05) between the experimental groups in the parameters of kinetics, plasma membrane and acrosome integrity. As for the potential of mitochondrial membrane, all treated groups were significantly (p≤0.05) larger than the control. It was concluded that the zinc oxide nanoparticles increase the mitochondrial membrane potential of ram sperm submitted to the freezing/thawing process.O objetivo deste estudo foi avaliar o efeito da adição de nanopartículas de óxido de zinco ao diluidor de criopreservação de sêmen ovino. Pools de sêmen (n=6), de três matrizes Santa Inês, foram diluídos em Tris-gema (5% glicerol), suplementado com nanopartículas de óxido de zinco (0, 25, 75 e 150μg/mL) na concentração de 200x106 espermatozóides/mL . As amostras foram congeladas em sistema automatizado e armazenadas em nitrogênio líquido (-196 °C). No momento da análise, as amostras de sêmen foram descongeladas (37 °C/30s) e avaliadas quanto à cinética espermática, integridade da membrana plasmática e acrossomal e potencial da membrana mitocondrial. Não houve diferença significativa (p&gt;0,05) entre os grupos experimentais nos parâmetros de cinética, membrana plasmática e integridade acrossômica. Quanto ao potencial de membrana mitocondrial, todos os grupos tratados foram significativamente (p≤0,05) maiores que o controle. Concluiu-se que as nanopartículas de óxido de zinco aumentam o potencial de membrana mitocondrial de espermatozóides ovinos submetidos ao processo de congelamento/descongelamento

Effect of Dietary Selenium and Vitamin E Supplementation on Testicular Morphology and Serum Testosterone Concentration in Goats Following Scrotal Insulation

Background: Heat directly applied to the testis has been providing information regarding the damage triggering mechanisms on spermatogenesis and possible treatments to prevent testicular changes. Testis submitted to heat-shock have inhibition of the local antioxidant defense mechanisms against lipid peroxidation and free radicals. Vitamin E and Selenium protect biological membranes against free radicals to prevent membrane lipid peroxidation. The current assay evaluated the effect of dietary supplementation with Selenium and Vitamin E on testicular parenchyma and testosterone levels of goats submitted to heat shock by scrotal insulation.Materials, Methods &amp; Results: The effect of dietary selenium and vitamin E supplementation on testicular parameters and serum testosterone concentration was evaluated in goats subjected to scrotal insulation. The animals were randomly allocated into two groups (n = 6) to receive either a control diet (CO) or a diet supplemented with selenium and vitamin E (SE). The animals received supplementation for 120 days: 60 days prior to scrotal insulation, 18 days during scrotal insulation and 42 days after scrotal insulation. Orchiectomy was performed on three animals from each group, immediately after the end of scrotal insulation. The remaining animals were neutered at the end of the experimental period (120 days). Testicles were routinely processed and embedded in glycol methacrylate, stained with toluidine blue/1% sodium borate and evaluated qualitative and quantitatively. Serum testosterone concentrations were determined by enzyme immunoassay at the time of the orchiectomy. Scrotal circumference was greater (P &lt; 0.05) in goats of the SE group (23.0 ± 1.00 cm) than those of the control group (20.0 ± 1.00 cm) at the end of the scrotal insulation period (Day 18).  At the end of the experimental period (Day 42 post-scrotal insulation (PSI)), the seminiferous tubule diameter and seminiferous epithelium height were greater (P &lt; 0.05) in the SE group than in control. Histological changes associated with testicular degeneration were detected after 18 days of scrotal insulation in the goats of the control group. The animals of SE group had some histological changes of seminiferous tubules but the majority of them had normal association of germ cells. Selenium and vitamin E supplementation did not seem to avoid testicular damage caused by scrotal insulation but accelerated testicular recovery after the removal of insulation. Testosterone serum levels were not changed in the animals submitted to scrotal insulation, with or without dietary supplementation with selenium and vitamin E.Discussion: In the current study, scrotal insulation for 18 days caused testicular degeneration in both groups. However, selenium and vitamin E supplementation were capable of maintaining the scrotal circumference on the 18th day of insulation in the SE group. Previous reports suggested that selenium and vitamin E could protect cell membranes against the harmful effects of reactive oxygen species. However, the histopathological changes and morphometric data observed in the both groups after 18 days of insulation demonstrated that supplementation with these antioxidants did not prevent the damage caused by heat stress.  In turn, at 42 days after the removal of insulation, the tubular diameter and seminiferous epithelium height was greater in animals supplemented with selenium and vitamin E. In addition, the animals that received supplementation had most of seminiferous tubules with cell associations of the seminiferous epithelium cycle. Vitamin E and selenium may reduce testicle sensitivity to heat and thereby shorten the spermatogenesis recovery time by 10 to 20 days. Selenium plus vitamin E added to feed was unable to prevent the degeneration of the testicular parenchyma in these animals. Nonetheless, the supplementation with both antioxidants hastened the recovery of spermatogenesis after the thermal injury

Cytotoxicity and genotoxicity assessment of the extract and lectins from Moringa oleifera Lam. Seeds / Avaliação da citotoxicidade e genotoxicidade do extrato e lectinas das sementes de Moringa oleifera Lam

Moringa oleifera seeds are used globally as a treatment for water and contain the lectins cMoL and WSMoL, which display coagulant activity. In this study, we sought to determine the cytotoxicity and genotoxicity of the M. oleifera seed extract (SE), prepared with the same procedure that people use for treating water, as well as cMoL and WSMoL, in human peripheral blood mononuclear cells (PBMCs). Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay, while genotoxicity was evaluated using the comet assay, with cell nucleoids categorized in classes ranging from 0 (without damage) to 4 (maximum damage). The PBMCs treated with SE, cMoL, and WSMoL displayed viability higher than 60% in treatments with concentrations up to 100 µg/mL. In addition, SE and cMoL displayed low genotoxicity owing to the detection of nucleoids in class 1. However, the number of nucleoids in all classes increased when 50 and 100 µg/mL of WSMoL was administered, reaching a damage frequency of 50.0%. Although M. oleifera SE, cMoL, and WSMoL were not cytotoxic to PBMCs after 24 h of exposure, dose-dependent genotoxic effects were observed, especially with WSMoL. These findings indicate that caution must be exercised when selecting a lectin/extract concentration for water treatment

Avaliação microbiológica do sêmen fresco e congelado de reprodutores caprinos

Objetivou-se avaliar a flora microbiana no sêmen fresco e congelado de reprodutores caprinos, assim como a eficácia dos antibióticos estreptomicina, penicilina e gentamicina, na viabilidade de doses de sêmen congeladas. Foram utilizados 25 reprodutores de diferentes raças, submetidos a duas colheitas de sêmen através do método da vagina artificial, após higiene da região prepucial. A primeira colheita do sêmen foi realizada visando o exame microbiológico e a segunda teve como objetivo proceder a congelação, após diluição em leite desnatado, utilizando penicilina + estreptomicina (A1), gentamicina (A2) ou sem antibiótico (A3). Ao proceder a avaliação microscópica no sêmen fresco, evidenciou-se média de 87,92 ± 7,76% de motilidade individual progressiva (MIP) e 4,96 ± 0,20 de vigor espermático. Em relação à avaliação bacteriana, constatou-se principalmente bactérias do gênero Staphylococcus spp e Bacillus sp. Após a congelação do sêmen, não foram evidenciadas diferenças (P&gt;;0,05) entre os grupos quanto a MIP e vigor espermático. Entretanto, na avaliação microbiológica pós-descongelação, a bactéria do gênero Staphylococcus spp esteve presente na maioria das amostras. Observou-se também que a gentamicina (13,3mg/mL) apresentou melhor atividade anti-microbiana no processo de congelação do sêmen, concluindo-se que pode ser o antibiótico usado na congelação do sêmen de reprodutores caprinos.The aim of this research was to evaluate the microbial flora in the fresh and frozen semen of goat reproducers, as well as the effectiveness of the antibiotics estreptomicin, penicillin and gentamicin in cryopreservation of semen. It were used 25 males of different breeds, submitted to two semen collect through the artificial vagina method after cleanliness of prepucial region. The first collection of semen aimed the microbiological exam. The second collection had as goal accomplish freezing, after dilution in skimmed milk, with penicillin + estreptomicin (A1), gentamicin (A2) or control (A3). After microscopic evaluation, it was evidenced average of 87.92 ± 7.76% of MIP and 4.96 ± 0.20 of spermatic vigor in fresh semen. Regarding bacterin evaluation, it was verified, mostly, bacteria of the gender Sthaphylococcus spp and Bacillus sp. After semen cryopreservation, it was observed that there wasn't difference (P&gt;;0.05) among groups in MIP and spermatic vigor. However, in the microbiological evaluation of frozen-thawed semen, bacteria of Sthaphylococcus spp gender was present in great part of samples. Gentamicin (13.3mg/mL) promoted larger inhibition of the bacterial growth in the semen post-freezing, concluding that gentamicin can be the antibiotic used for freezing of goat semen

Renovação parcial do meio de cultura como perspectiva de aumentar a eficiência de um sistema de cocultoura de embriões sem fluxo contínuo de CO2 Partial replacement of the culture medium in order to enhance a embryos coculture system without continuous CO2 flow

Substituindo-se parcialmente (30%) os meios de cultura TC M 199/NaHCO3 (199/NaHCO3) e TC M 199/25mM HEPES (199/HEPES) a intervalos de 24, 48 ou 72 horas, testou-se a possibilidade de incrementar a obtenção de blastocistos expandidos (BLE) a partir de embriões Mus musculus no estádio de duas células incubados sem fluxo contínuo de CO2. Após distribuição aleatória dos embriões em tubos de ensaio contendo 2ml de meio de cultura e monocamada celular de oviduto bovino, os referidos tubos foram hermeticamente fechados e colocados em estufa bacteriológica a 37&deg;C durante 96 horas. A renovação do meio de cultura em qualquer período não incrementou a porcentagem de embriões que alcançou o estádio de BLE, todavia, o número total de BLE obtido com o 199/HEPES (79,0%) foi superior (P &pound; 0,01) ao verificado com o 199/NaHCO3 (65,0%).It has been evaluated, in a coculture system without continuous CO2 flow, the possibility to enhance the percentage of blastocists obtained from two cells mouse embryos by partial (30%) replacement (each 24, 48 and 72 hours of intervals) of two culture media (TCM 199/NaHCO3 and TCM 199/25 mM HEPES). The embryos, randomiy allocated in closed cultures tubes with 2 ml of medium and bovine oviduct monolayer, were maintained at 37&deg;C during 96 hours. No diferences between spreaded blastocists were verifica when the media were replaced at ali intervals, however, the medium TCM 199/25mM HEPES was significantiy more effective (P &pound; 0.01) then TCM 199/NaHCO3