This study assessed the effect of flow cytometry sexing on sperm longevity and the capacity of sperm to bind to oviductal cells. Each ejaculate from four bulls was divided into two fractions: the first was immediately frozen as non sexed sperm (NS) and the second was sexed originating X-and Y-bearing sperm. The fourth treatment had sex-sorted X and Y sperm (XY) combined. Sperm from each group was assessed for sperm characteristics after thawing, after washing and at 2, 4, 8 and 12 h of incubation at 39°C in 5% CO 2 in air. For the binding test, sperm were incubated in sp-TALP medium for 30 min or 24 h with oviductal explants. Percentages of motility (58.1 ± 4.3 and 35.2 ± 4.4), progressive motility (46.1 ± 6.1 and 25.7 ± 4.8), mitochondrial membrane potential (79.2 ± 8.3 and 69.0 ± 6.3), plasma membrane stability (77.4 ± 4.6 and 19.4 ± 4.2), and live sperm with intact acrosome (57.2 ± 8.5 and 31.3 ± 7.9) were higher in NS than in XY, respectively (P < 0.05). Those differences were maintained for up to 8 h. The sexing process did not affect the sperm binding to the explants after 30 min. However, after 24 h, XY had less (6.7 ± 2.0) sperm bound to explants than NS (23.6 ± 7.2). In conclusion, even though XY was of lower quality than NS, the decreases in quality in both NS and XY groups were similar between groups during incubation. Moreover, the sex-sorting process affected the ability of sperm to remain bound to oviductal explants.

A variety of laboratory tests were developed to obtain more reliable results of sperm evaluation and increase the accuracy of sperm fertility predictions. These tests detected damage of sperm specific compartments or organelles, which cannot be detected in routine sperm analysis. The use of fluorescent probes and detection using fluorescent microscopy or flow cytometry is an important tool but a more precise and accurate laboratory test is needed. Propidium iodide and 6-carboxyfluorescein diacetate are used for evaluations of plasmatic membrane integrity. Fluorescein isothiocyanate, associated with conjugated lecithin Psium sativum or Arachis hypogaea, are used for evaluations of acrosome integrity. Two probes, MitoTracker or Rhodamine123, are generally used to measure the absence or presence of mitochondrial potential. However, a better option is 5,5'; 6,6' -tetrachloro -1,1'; 3,3' -tetraetilbenzimidazolil-carbocyanine (JC-1) dye, which assesses not only the presence of mitochondrial potential and distinguished spermatozoa with poorly and highly functional mitochondria. Two techniques, TUNEL or COMETA, and the Acridine Orange Test (AOT) dye are used to evaluate chromatin integrity. A fluorescence technique based on chlortetracycline (CTC) or Merocyanine 540 is used to estimate whether sperm pass by or through the capacitation process. This review focuses on the fluorescent probes that are most widely used to evaluate plasma membrane integrity, capacitation, acrosome integrity, chromatin integrity and mitochondrial potential. Key words: Fluorescence microscopy, sperm, cryopreservation ResumoDiversos testes laboratoriais têm sido desenvolvidos com o intuito de obter resultados confiáveis nas avaliações espermáticas, aumentando a qualidade e a confiabilidade do sêmen utilizado em técnicas de reprodução assistida. Esses testes têm permitido detectar danos em compartimentos e organelas específicas da célula espermática, que não são detectados nas análises de rotina. Dentre esses testes, o uso de sondas fluorescentes e sua detecção em microscópio de epifluorescência ou citometria de fluxo se tornaram ferramenta importante quando uma avaliação mais acurada é necessária. Para a avaliação da integridade de membrana plasmática, pode ser utilizado o iodeto de propídio, associado ao diacetato de 6-carboxifluoresceína. As avaliações de integridade acrossomal podem ser feitas através do isotiocianato de fluoresceína, associado às lecitinas conjugadas, como a Psium sativum ou Arachis hypogaea. O potencial mitocondrial pode ser avaliado quanto à ausência ou presença através da sonda Mitotracker ou Rodamina 123. Outra opção ainda melhor pode ser observada utilizando o corante iodeto de 5,5'; 6,6' -tetracloro -1,1'; 3,3' -tetraetilbenzimidazolil-carbocianina (JC-1), este corante avalia a presença do potencial mitocondrial e avalia quanto à classificação do grau. Para avaliar a integridade

Summary: Reproductive biotechnologies such as cooling and freezing semen are employed in order to obtain genetic enhancement in domestic animal breeds. However, cryopreservation of donkey semen, as in several other domestic mammalian species, has not yet reached a standard procedure that provides satisfactory and repeatable results, as in bulls. Despite there are over 43 million donkeys worldwide, there are only few studies about donkey semen preservation. The aim of this study was to evaluate the predictive potential of hypo-osmotic test and the supra vital staining in raw semen to be used as indicators for donkey semen freezability. Ejaculates were collected twice per week from 10 fertile donkey stallions. Volume, appearance (color and density), concentration, total motility, morphology, sperm viability (supra vital staining -eosin-nigrosin-EN) and plasma membrane integrity (hypo-osmotic swelling test -HOST) were evaluated in raw semen. After dilution the samples were centrifugated and sperm pellets were resuspended in a freezing extender to a concentration of 50 ×10 6 cells/mL. Aliquots were packed into 0.5 mL straws and placed in the refrigerator (5°C) for 20 min. Subsequently these straws were kept above liquid nitrogen for 20 min, then plunged into nitrogen and stored in a holding tank. Post-thaw analyzes consisted in computerized analysis of sperm movement characteristics, sperm viability (EN), plasma membrane integrity (HOST and fluorescent probe) and acrosome membrane integrity using fluorescent probe. High correlation was found between EN (0.93) and HOST (0.69) in raw semen when compared with total motility after frozen-thawed semen. Low correlation was found between EN and HOST when compared with plasma and acrosomal membrane integrity in post-thawed with fluorescent probes. Since sperm motility is one of the key parameters to evaluate the quality of frozen semen, hypo-osmotic test and supra vital staining can be considered good predictive indicators in raw semen for donkey semen freezability.Keywords: Equus Asinus, cryopreservation, eosin-nigrosin, plasma membrane integrity, semen, reproduction Citation: Arruda de Oliveira R., Batista Silva Teixeira A., Almeida Pignataro T., Freitas M. L., Castro Alves Teixeira H., de Oliveira Carvalho J., Alves Nune Dode M., Pivato I., Budik S. (2017) Evaluation of Hypo-osmotic swelling test and supra vital staining technique as indicators for donkey semen freezability. Pferdeheilkunde 33,[159][160][161][162][163][164]

Resumo -O objetivo deste estudo foi comparar a cinética de sêmen bovino criopreservado não sexado, sexado X e sexado Y antes e depois da seleção espermática por gradiente de Percoll. Amostras criopreservadas de sêmen não sexado (grupo NS) e sexado X (grupo SX) e Y (grupo SY) por citometria de fl uxo, de quatro touros, foram avaliadas quanto à motilidade e à cinética espermática com o "computer-assisted semen analysis" (CASA) e o restante da amostra de cada grupo foi submetido à seleção espermática em gradiente de Percoll (45:60%). Após a seleção, foram realizadas as mesmas avaliações que antes da passagem pelo Percoll. A motilidade do grupo NS foi superior à dos grupos SX e SY e não foi observada diferença entre os grupos SX e SY nos parâmetros de cinética espermática obtidos pelo CASA, antes ou após a passagem pelo Percoll. Foi observado aumento na motilidade para todos os grupos como efeito da seleção pelo Percoll. O processo de sexagem por citometria de fl uxo afeta a cinética espermática, e a passagem pelo Percoll aumenta a motilidade do sêmen sexado e não sexado sem alterar a cinética do sêmen não sexado.Termos para indexação: gradiente de Percoll, motilidade espermática, sêmen sexado. Kinetics of bovine cryopreserved sperm cells after sexing by fl ow cytometryAbstract -The objective of this study was to compare the kinetics of bovine cryopreserved nonsexed, sexed for X and sexed for Y sperm cells before and after selection using Percoll gradient. Cryopreserved nonsexed (NS group) semen samples and sexed for X (SX group) and for Y (SY group) by fl ow cytometry, from four different sires, were analyzed for motility by computer-assisted semen analysis (CASA), and the remainder of the sperm samples for each group was submitted to Percoll (45:60%) gradient selection. After Percoll selection, the same sperm analyses done before the passing through Percoll gradient were performed. Sperm motility was higher in the NS group than in the SX and SY groups, and no differences were observed between these last two groups for any of the sperm parameters evaluated by CASA, either before or after Percoll selection. Percoll increased motility for all groups. Changes in other characteristics evaluated by CASA were observed only in the NS group. Sex-sorting procedure by fl ow cytometry affected sperm kinetics, and Percoll gradient selection increases motility in sexed and nonsexed sperm without affecting the kinetics of nonsexed sperm.

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