From Past to Present: The Link Between Reactive Oxygen Species in Sperm and Male Infertility

Reactive oxygen species (ROS) can be generated in mammalian cells via both enzymatic and non-enzymatic mechanisms. In sperm cells, while ROS may function as signalling molecules for some physiological pathways, the oxidative stress arising from the ubiquitous production of these compounds has been implicated in the pathogenesis of male infertility. In vitro studies have undoubtedly shown that spermatozoa are indeed susceptible to free radicals. However, many reports correlating ROS with sperm function impairment are based on an oxidative stress scenario created in vitro, lacking a more concrete observation of the real capacity of sperm in the production of ROS. Furthermore, sample contamination by leukocytes and the drawbacks of many dyes and techniques used to measure ROS also greatly impact the reliability of most studies in this field. Therefore, in addition to a careful scrutiny of the data already available, many aspects of the relationship between ROS and sperm physiopathology are still in need of further controlled and solid experiments before any definitive conclusions are drawn

Comparação ente dois métodos de inseminação artificial utilizando sêmem congelado em gatos domésticos(Felis catus)

O objetivo deste estudo foi comparar a eficiência das técnicas de inseminação artificial intra-vaginal (IAIV) e intra-uterina (IAIU) com sêmen congelado/descongelado em gatos domésticos através da taxa de prenhez. O ejaculado de dois gatos adultos foi colhido com vagina artificial e o sêmen avaliado para motilidade (CASA), morfologia espermática e integridade de membrana plasmática. Após diluição em meio TRIS/OEP (4% de glicerol), as amostras foram envasadas em palhetas francesas de 0,25 mL (25 x 106 de espermatozóides móveis) as quais, após 20 minutos à 5ºC, permaneceram por 15 minutos em vapor de nitrogênio líquido, sendo posteriormente mergulhadas. Para cada IA, quatro palhetas do mesmo gato foram descongeladas por 12 segundos à 46ºC e centrifugadas a 250xg por 8 minutos. O pellet ressuspendido em 100 æL do meio foi analisado como descrito anteriormente. Nas gatas, o estro e a ovulação foram induzidos com 100 UI de eCG e, após 84 horas, 100 UI de hCG. Após 30 horas da indução da ovulação, as gatas foram inseminadas pelo método intra-uterino ou intra-vaginal, com o sêmen obtido do gato A ou B (n=8 gatas para cada método de IA). Para a análise estatística os testes de Tukey e de Wilcoxon foram utilizados para as variáveis de qualidade espermática, enquanto o teste de Fisher foi usado para comparar os métodos de IA, estabelecendo diferença significativa quando p<0,05. Embora tenha sido observada uma queda acentuada de motilidade, integridade de acrossomo e integridade de membrana plasmática nas amostras seminais descongeladas de ambos os gatos, um índice de prenhez de 75% foi alcançado quando utilizado o método de IA intra-uterino, contra 0% com o método intra-vaginal.The aim of this study was to compare the efficiency of the intravaginal and the intrauterine artificial insemination methods using domestic cat frozen/thawed semen by determining the pregnancy rate. The sperm was collected from two tom cats using an artificial vagina and the samples were assessed for motility (CASA), sperm morphology and plasma membrane integrity. After dilution with TRIS/OEP (4% of glycerol), the sperm samples were loaded into 0.25 mL straws (25 x 106 of sperm cells presenting motility) which, after 20 minutes under 5ºC, were placed in liquid nitrogen vapour for 15 minutes, being afterwards immersed. For each AI, four straws from the same male were thawed during 12 seconds at 46ºC and centrifuged at 250xg for 8 minutes. The pellet was ressuspended in 100 æL of the extender and analyzed as described above. In the queens, estrus and ovulation were induced using 100 UI of eCG and, after 84 hours, 100 UI of hCG. After 30 hours from the ovulation induction, the females were inseminated by the intrauterine or by the intravaginal methods, using the semen obtained by male A or B (n=8 females for each AI method). For statistical analysis the Tukey test and the Wilcoxon test were used for the sperm quality results, while the Fisher test was used to compare the two AI methods, with p<0.05 taken as significant. Although a pronounced decrease in motility, acrosome integrity and plasma membrane integrity were observed for the sperm samples from both cats, a pregnancy rate of 75% was achieved when using the intrauterine AI method, against 0% with the intravaginal AI method.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

COMPARISON BETWEEN TWO METHODS OF STAINING FOR ASSESSMENT OF MORPHOLOGY AND ACROSOME IN DOMESTIC CAT (Felis catus) SPERMATOZOA COMPARAÇÃO ENTRE DOIS MÉTODOS DE COLORAÇÃO PARA ANÁLISE MORFOLÓGICA E ACROSSOMAL DE ESPERMATOZÓIDES DE GATO DOMÉSTICO (Felis catus).

&lt;span&gt;&lt;font size="3"&gt;&lt;font face="Times New Roman"&gt;&lt;span&gt;&lt;p align="justify"&gt;The objective of this study was to evaluate the efficiency of the modified Karras staining technique (KA) to analyze domestic cat sperm morphology by comparing it with the Fast Green FCF/ Rose Bengal staining (FR), previously used for this species. Four adult cats were used, from which sperm samples were collected four times in alternate days for each tom using an artificial vagina (n=16 ejaculates). Both staining techniques were performed for each ejaculate. For the FR staining technique, the semen &lt;em&gt;in natura&lt;/em&gt; was diluted in 2.9% sodium citrate and, afterwards, in the staining solution. After 70 seconds, smears were made onto slide and dried at 37ºC. For the KA staining technique, previously made and formol saline fixed slides were sequentially immersed in Rose Bengal solution, Tannin&lt;font size="2" color="#ff0000"&gt;&lt;font size="3" color="#ff0000"&gt; &lt;/font&gt;&lt;/font&gt;&lt;font size="3"&gt;solution, and Victoria Blue B solution, and dried at room temperature. For sperm evaluation, 200 sperm cells were assessed for each staining technique in all ejaculate samples using a bright field microscope at 1000X magnification. Statistical &lt;span&gt;analysis used the non-parametric Wilcoxon test, establishing significance at p&lt;0.05. For the KA staining technique, higher percentage of distal cytoplasmic droplets and lower percentage of sperm head defects were obtained when compared to the FR staining technique. This way, both staining techniques were not totally efficient for the assessment of morphological defects found in the domestic cat &lt;em&gt;in natura &lt;/em&gt;spermatozoa.&lt;/span&gt;&lt;/font&gt;&lt;/p&gt;&lt;font size="3"&gt;&lt;span&gt;&lt;span&gt;&lt;p align="justify"&gt;KEY WORDS: Acrosome, domestic cat, spermatozoa, sperm morphology, staining.&lt;/p&gt;&lt;/span&gt;&lt;/span&gt;&lt;/font&gt;&lt;/span&gt;&lt;/font&gt;&lt;/font&gt;&lt;/span&gt;&lt;p&gt; &lt;/p&gt; &lt;font size="3"&gt;&lt;font face="Times New Roman"&gt;&lt;span style="color: black"&gt;&lt;span&gt;&lt;p align="justify"&gt;O objetivo do estudo foi avaliar a eficiência do método de coloração Karras modificado (KA) para a análise da morfologia espermática no gato doméstico através da comparação com a coloração Fast Green FCF/Rosa Bengala (FR), previamente utilizada para esta espécie. Utilizaram-se quatro gatos adultos, colhendo-se quatro vezes amostras de sêmen em dias alternados para cada animal através de vagina artificial (n=16 ejaculados). Para cada ejaculado, realizaram-se duas colorações. Para a coloração FR, o sêmen &lt;em&gt;in natura&lt;/em&gt; foi diluído em citrato de sódio 2,9% e, posteriormente, em solução de coloração. Após setenta segundos, procedeu-se a esfregaços em lâminas, as quais foram secas a 37ºC. Para a coloração KA, os esfregaços previamente confeccionados e fixados em formol salino foram imersos seqüencialmente nas soluções de Rosa Bengala, Tanino e Azul Vitória e secas em temperatura ambiente. Avaliaram-se duzentas células para cada tipo de coloração em todos os ejaculados, usando-se microscópio de luz em aumento de 1.000X. Efetuou-se análise estatística mediante o teste não-paramétrico de Wilcoxon, estabelecendo diferença significativa quando p&lt;0,05. Para a coloração de KA, obtiveram-se maior porcentagem de gota citoplasmática distal e menor porcentagem de defeitos de cabeça quando comparada à coloração FR. Assim, nenhuma das colorações mostrou-se totalmente eficiente na identificação dos defeitos de morfologia encontrados na avaliação do sêmen &lt;em&gt;in natura&lt;/em&gt; de gatos domésticos.&lt;/p&gt;&lt;span&gt;&lt;p align="justify"&gt;PALAVRAS-CHAVES: Acrossomo, coloração, espermatozóide, gato doméstico, morfologia espermática.&lt;/p&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/font&gt;&lt;/font&gt;&lt;p&gt; &lt;/p&gt

Nonpregnant and pregnant adult female rats affected by maternal diabetes environment

Maternal diabetes-mediated fetal programming is widely discussed, however, it is important to define the extent to which intrauterine hyperglycemia interferes with the health of female pups, along with determining whether these changes can be perpetuated across generations. This study aimed to evaluate the effects of maternal diabetes on fetal programming and the repercussions on the metabolism of pregnant and nonpregnant female pups. Diabetes status was induced (diabetic group—D) using streptozotocin (a beta cell cytotoxic drug) on the fifth postnatal day of female rats, while controls received a citrate buffer (Control—C). In adulthood, the rats were mated to obtain their female pups. At 90 days of age, half of the female pups were mated (preg) and the other half continued virgin (Npreg). Furthermore, they were distributed into four groups: OC/Npreg and OC/preg—female pups from control mothers; OD/Npreg and OD/preg—female pups from diabetic mothers. At 115 days of life and/or 17 days of pregnancy, the oral glucose tolerance test (OGTT) was performed with blood collection for insulin measurement. At 120 days of life and/or 21 days of pregnancy, the rats were anesthetized and euthanized to determine their blood oxidative stress status. The OD/Npreg group showed glucose intolerance during OGTT (p p p = 0.0027). An increase in homeostatic model assessment β was shown in the pregnant groups, regardless of maternal diabetes (p p p = 0.0005) and decreased superoxide dismutase activity (p = 0.0063). Additionally, small fetuses for gestational age (p < 0.0001) were found in these rats. In conclusion, exposure to maternal hyperglycemia compromises the glycemic metabolism of female pups before and during pregnancy and causes oxidative stress, IR, and impaired fetal growth during pregnancy.</p