Efficient, fast and low-cost strategies for DNA extraction from different nucleated sheep cells

DNA extraction is usually the first step to perform molecular studies. This process can be nonviable due to genomic DNA (gDNA) extraction commercial kits prices. Furthermore, available DNA extraction protocols generally have high specificity, limiting their use to specific sources of biological material. In order to reduce costs, optimize time and laboratory logistics, besides to demonstrate a versatile protocol, the present study worked on an efficient DNA extraction protocol from somatic and non-somatic cells, using biological material from sheep as a model. For that, gDNA was extracted from whole blood, spermatozoa, and hair bulb cells, collected from three adult sheep, transported at 5ºC and stored at -20ºC until lab procedures. After extraction, gDNA concentration and purity were evaluated in a nano spectrophotometer. gDNA concentration from whole blood was greater (p < 0.05) than extracted from hair bulb cells, which in turn was superior (p < 0.05) than in spermatozoa. Also, gDNA from whole blood and, followed by, sperm showed greater (p < 0.05) purity when compared to gDNA of hair bulb cells. Adapting a gDNA extraction protocol, originally developed for bovine whole blood, enabled to obtain and isolate gDNA in different nucleated sheep cells.(AU)

Functional assessment of diluent choice for semen cryopreservation from stallions with high and low freezability

Background: Fertility using horse frozen-thawed semen remains lower than in other livestock species. This fact suggeststhat horse semen hold intrinsic sensitivity to cryoinjury that must be investigated. Moreover, there is substantial evidenceof genetic factors upon horse cryopreservation outcome. Nonetheless, diluent and cryoprotectant choice for horse semencryopreservation are under intense research. Thus these factors could be explored to identify conditions that may increasesemen viability after thawing. The aim of this work was to evaluate the effect of diluents Botu-Crio®,Lactose-EDTA®, andINRA-82® on cryopreserved semen from stallions with high (HFA) and low freezability (LFA).Materials, Methods & Results: Frozen-thawed semen was evaluated for motility and membrane integrity using computerassisted semen analysis (CASA), and also inferred for sperm DNA fragmentation by sperm chromatin structure assay during the thermoresistance test (TRT). Comparisons for each parameter were done in a pair-wise fashion between HFA andLFA semen at one-hour intervals during the TRT (0 h - 4 h). Sperm motility in HFA, regardless of the diluent, was larger(P 0.05)at 0 h and 3 h. Sperm DNA fragmentation was lower (P < 0.05) in HFA semen at 0 h and 1 h.Discussion: Artificial insemination in horses using frozen-thawed semen is gaining wider acceptance under commercialsettings, although its current limited outreach due to low semen viability after thawing. Therefore, several efforts weremade toward ...

Functional assessment of diluent choice for semen cryopreservation from stallions with high and low freezability

Background: Fertility using horse frozen-thawed semen remains lower than in other livestock species. This fact suggeststhat horse semen hold intrinsic sensitivity to cryoinjury that must be investigated. Moreover, there is substantial evidenceof genetic factors upon horse cryopreservation outcome. Nonetheless, diluent and cryoprotectant choice for horse semencryopreservation are under intense research. Thus these factors could be explored to identify conditions that may increasesemen viability after thawing. The aim of this work was to evaluate the effect of diluents Botu-Crio®,Lactose-EDTA®, andINRA-82® on cryopreserved semen from stallions with high (HFA) and low freezability (LFA).Materials, Methods & Results: Frozen-thawed semen was evaluated for motility and membrane integrity using computerassisted semen analysis (CASA), and also inferred for sperm DNA fragmentation by sperm chromatin structure assay during the thermoresistance test (TRT). Comparisons for each parameter were done in a pair-wise fashion between HFA andLFA semen at one-hour intervals during the TRT (0 h - 4 h). Sperm motility in HFA, regardless of the diluent, was larger(P < 0.05) than LFA, both on 0h and 1h. In the 2h evaluation, sperm motility using Botu-Crio® and Lactose-EDTA® wasgreater (P < 0.05) for HFA. Analysis of sperm membrane integrity was similar between HFA and LFA semen (P > 0.05)at 0 h and 3 h. Sperm DNA fragmentation was lower (P < 0.05) in HFA semen at 0 h and 1 h.Discussion: Artificial insemination in horses using frozen-thawed semen is gaining wider acceptance under commercialsettings, although its current limited outreach due to low semen viability after thawing. Therefore, several efforts weremade toward ... (AU)

Influence of male to female ratio on hormone profiles and reproductive performance of anestrus postpartum ewes subjected to the male effect

Background: Complete isolation of genders allows intense estrous induction and synchronization once rams are introduced in ewe flocks at the onset of the breeding season (BS). This management practice, defined as the male effect, results from a neuroendocrine process mediated by pheromones. The male effect is a straightforward procedure to induce estrous in noncycling ewes, but conditions for its use have not been fully explored. Thus, this study aimed to evaluate hormone levels and ovarian activity of postpartum ewes in anestrus which are subjected to the male effect under different male to female ratios. Material, Methods & Results: Pospartum females were selected according to body condition score and cyclicity status. Females were kept apart from males during 30 days at a distance of 10 m. Anestrus and ovulation were determined by P4 measures on days 10, 20 and 30 after isolation from males. After P4 concentration diagnosis, anestrus ewe (n = 99) were subjected to male to ewe ratios (MFR) of 1:20 (MFR20), 1:30 (MFR30) and 1:40 (MFR40). Santa Inês rams (n = 3) of proven fertility were used. Three females of each group were randomly subjected to blood collection for LH concentration analysis. Ovarian activity was performed by ultrasonography after estrus manifestation in six ewe of each group. Estrus events were observed twice a day during the BS of 35 days, and estrus [...]

Influence of male to female ratio on hormone profiles and reproductive performance of anestrus postpartum ewes subjected to the male effect

Background: Complete isolation of genders allows intense estrous induction and synchronization once rams are introduced in ewe flocks at the onset of the breeding season (BS). This management practice, defined as the male effect, results from a neuroendocrine process mediated by pheromones. The male effect is a straightforward procedure to induce estrous in noncycling ewes, but conditions for its use have not been fully explored. Thus, this study aimed to evaluate hormone levels and ovarian activity of postpartum ewes in anestrus which are subjected to the male effect under different male to female ratios. Material, Methods & Results: Pospartum females were selected according to body condition score and cyclicity status. Females were kept apart from males during 30 days at a distance of 10 m. Anestrus and ovulation were determined by P4 measures on days 10, 20 and 30 after isolation from males. After P4 concentration diagnosis, anestrus ewe (n = 99) were subjected to male to ewe ratios (MFR) of 1:20 (MFR20), 1:30 (MFR30) and 1:40 (MFR40). Santa Inês rams (n = 3) of proven fertility were used. Three females of each group were randomly subjected to blood collection for LH concentration analysis. Ovarian activity was performed by ultrasonography after estrus manifestation in six ewe of each group. Estrus events were observed twice a day during the BS of 35 days, and estrus [...](AU)

Lipid structural information from a single equine embryo by MALDI-MS / Informação estrutural de lipídios a partir de um único embrião equino pela técnica de MALDI-MS

It was reported the potential of MALDI-MS for the characterization of lipid species present in a single equine embryo, and studied some lipid structures detected by collision induced dissociation (CID) experiments. In the positive ion mode spectrum, it were observed mostly protonated and sodiated species of sphingomyelins (SM), phosphatidylcholines (PC) and triacylglycerols (TAG). In the negative ion mode, it were observed phosphatidylethanolamines (PE) and phosphatidylinositols (PI). MS/MS spectrum of most intense lipid ions was performed to show MALDI-MS/MS structural information potential. MS/MS spectrum in the positive mode of m/z 760.6 (attributed as PC34:1) depicted characteristic PC fragments of m/z 184.1 (choline polar head), and the neutral loss (NL) of 183 (phosphorylcholine). For the ion of m/z 766.6 (attributed as PE 38:5), we observed the NL of 140, characteristic of PE. For the ion of m/z 808.7 (attributed as PC 38.5), besides the fragment at m/z 184.1 at the NL of 183, it was possible to observe the loss of trimethylamine (ion of m/z 749.6), and the cyclophosphane (ion of m/z 147.0). Finally, for the negative ion mode, we isolated and fragmented the ion at m/z 863.6, which was attributed as PI 36:1 due to the presence of m/z 153 (glycerol phosphate – H2 O-H), 223 (phospho inositol – 2H2 O-H), 241 (phospho inositol – H2 O-H), 281 (oleic acid), and 581.3 (lysophosphoinositol – H2 O-H). It was concluded that MALDI-MS allowed the detection of a broad range of PC, SM, PE, PI and TAG lipid species, as well as a fast and confident characterization of lipid structures from a single equine embryo.

É relatado o potencial da técnica de MALDI-MS para caracterizar espécies de lipídios presentes em um único embrião equino e estudadas algumas estruturas lipídicas detectadas por dissociação induzida por colisão (CID). No espectro de modo íon positivo, foram observadas espécies, principalmente, protonadas e sodiadas de esfingomielinas (SM), fosfatidileolinas (PC) e triacilgliceróis (TAG). No modo negativo, foram observadas fosfatidiletanolaminas (PE) e fosfatidilinositos (PI). Espectros de íons de lípidos com maior intensidade foram utilizados para demonstrar o potencial da informação estrutural por MALDI-MS/MS. O espectro no modo positivo de m/z (massa sobre carga) 760,6 (atribuída como PC34:1) apresentou características de fragmentos PC de m/z 184,1 (denominada cabeça polar de colina), além de perda neutral (NL) de m/z 183 (fosforilcolina). Para o íon de m/z 766,6 (atribuída como PE38:5), observou-se a NL de 140, característica do PE. Para o íon de m/z 808,7 (38,5 atribuído como PC), além do fragmento m/z 184,1 na NL de 183, foi possível observar a perda de trimetilamina (íon de m/z 749,6) e o ciclofosfano (íon de m/z 147,0). Finalmente, para o modo de íon negativo, foram isolados e fragmentados o íon de m/z 863,6 que foi atribuído como PI36:1, devido à presença de m/z 153 (fosfato de glicerol – H2 O-H ), 223 (inositol fosfo - 2H2 O-H) , 241 (fosfoinositol – H2 O-H), 281 (ácido oleico) e 581,3 (lisofosfoinositol – H2 O+H). Foi concluído que a MALDI - MS permite a detecção de uma ampla gama de espécies de PC, SM, PE, PI e TAG lipídicas, bem como a caracterização rápida e confiante de estruturas lipídicas a partir de um único embrião equino.

Lipid structural information from a single equine embryo by MALDI-MS / Informação estrutural de lipídios a partir de um único embrião equino pela técnica de MALDI-MS

It was reported the potential of MALDI-MS for the characterization of lipid species present in a single equine embryo, and studied some lipid structures detected by collision induced dissociation (CID) experiments. In the positive ion mode spectrum, it were observed mostly protonated and sodiated species of sphingomyelins (SM), phosphatidylcholines (PC) and triacylglycerols (TAG). In the negative ion mode, it were observed phosphatidylethanolamines (PE) and phosphatidylinositols (PI). MS/MS spectrum of most intense lipid ions was performed to show MALDI-MS/MS structural information potential. MS/MS spectrum in the positive mode of m/z 760.6 (attributed as PC34:1) depicted characteristic PC fragments of m/z 184.1 (choline polar head), and the neutral loss (NL) of 183 (phosphorylcholine). For the ion of m/z 766.6 (attributed as PE 38:5), we observed the NL of 140, characteristic of PE. For the ion of m/z 808.7 (attributed as PC 38.5), besides the fragment at m/z 184.1 at the NL of 183, it was possible to observe the loss of trimethylamine (ion of m/z 749.6), and the cyclophosphane (ion of m/z 147.0). Finally, for the negative ion mode, we isolated and fragmented the ion at m/z 863.6, which was attributed as PI 36:1 due to the presence of m/z 153 (glycerol phosphate H2 O-H), 223 (phospho inositol 2H2 O-H), 241 (phospho inositol H2 O-H), 281 (oleic acid), and 581.3 (lysophosphoinositol H2 O-H). It was concluded that MALDI-MS allowed the detection of a broad range of PC, SM, PE, PI and TAG lipid species, as well as a fast and confident characterization of lipid structures from a single equine embryo.(AU)

É relatado o potencial da técnica de MALDI-MS para caracterizar espécies de lipídios presentes em um único embrião equino e estudadas algumas estruturas lipídicas detectadas por dissociação induzida por colisão (CID). No espectro de modo íon positivo, foram observadas espécies, principalmente, protonadas e sodiadas de esfingomielinas (SM), fosfatidileolinas (PC) e triacilgliceróis (TAG). No modo negativo, foram observadas fosfatidiletanolaminas (PE) e fosfatidilinositos (PI). Espectros de íons de lípidos com maior intensidade foram utilizados para demonstrar o potencial da informação estrutural por MALDI-MS/MS. O espectro no modo positivo de m/z (massa sobre carga) 760,6 (atribuída como PC34:1) apresentou características de fragmentos PC de m/z 184,1 (denominada cabeça polar de colina), além de perda neutral (NL) de m/z 183 (fosforilcolina). Para o íon de m/z 766,6 (atribuída como PE38:5), observou-se a NL de 140, característica do PE. Para o íon de m/z 808,7 (38,5 atribuído como PC), além do fragmento m/z 184,1 na NL de 183, foi possível observar a perda de trimetilamina (íon de m/z 749,6) e o ciclofosfano (íon de m/z 147,0). Finalmente, para o modo de íon negativo, foram isolados e fragmentados o íon de m/z 863,6 que foi atribuído como PI36:1, devido à presença de m/z 153 (fosfato de glicerol H2 O-H ), 223 (inositol fosfo - 2H2 O-H) , 241 (fosfoinositol H2 O-H), 281 (ácido oleico) e 581,3 (lisofosfoinositol H2 O+H). Foi concluído que a MALDI - MS permite a detecção de uma ampla gama de espécies de PC, SM, PE, PI e TAG lipídicas, bem como a caracterização rápida e confiante de estruturas lipídicas a partir de um único embrião equino.(AU)

Avaliação física de equinos da cavalaria do estado de Sergipe / Equine physical evaluation cavalry Sergipe state / Evaliacion física equina estado caballería Sergipe

Este estudio tuvo como meta evaluar los caballos de competición y de umbral aeróbico Montada Escuadrón de Caballería de Ia Policía dei Estado de Sergipe, que trabajan en Ias actividades de baja intensidad media. Quince caballos, mestizas edades de ocho a dieciocho anos, fueron sometidos a Ia prueba de esfuerzo decampo, con una duración de 20 minutos divididos en seis velocidades (de 2,5 a 9 rn/s). Se recogieron durante y después de Ia prueba Ias muestras para Ia medición de lactato deshidrogenasa (LDH) (reposo y una velocidad de O,5,09,02,5 m 1 s después de 30 minutos), Ia creatina quinasa (CK) y aspartato aminotransferasa (AST) (reposo ydespués de 30 minutos, 12 y 14 horas). Previa y después se realizaron los exámenes de Ias pruebas de resistenciade broncoscopia. En endoscopio examen, esfuerzo previa a Ia prueba, se observó Ia presencia de cambios en elsistema respiratorio en eI46,66% (7/15) de los caballos hay dos casos de hemiplejía laríngea y Dorsal desplazamientodei paladar blando, el cambio etmoidal, el cáncer de Ia epiglotis, Ia neoplasia osteo faringeana y Ias secreciones bronquiales. Se concluyó que los caballos de Ia patrulla de Ia policía montan Aracaju, son capaces de lIevar a caboactividades de patrulla montada, por 10 que Ia prueba de estrés asociado con Ia medición de los niveles sé ricos deCK, AST Y LDH son herramientas importantes para Ia determinación dei condicionamiento físico.

This study aimed to evaluate the performance and aerobic threshold horses Mounted Police Squadronof Cavalry of the State of Sergipe, who work in the average intensity low activities. Fifteen horses, crossbred ageseight to eighteen, were subjected to the stress test field, lasting 20 minutes divided into six speeds (from 2,5 to 9 mls). During and post-test samples were collected for measurement of lactate dehydrogenase (LDH) (rest and speedsof 0,5,0,9,0,2,5 m/s after 30 minutes), creatine kinase (CK) and aspartate aminotransferase (AST) (rest and after 30minutes, 12 and 14 hours). Preview and then the stress test bronchoscopy examinations were performed. Onexamination endoscope, pre-test effort, it was observed the presence of changes in the respiratory system in 46.66%(7/15) of horses there two cases of laryngeal hemiplegia and one dorsal displacement of the soft palate, ethmoidchange, epiglottis neoplasia, osteofaringeana neoplasia and bronchial secretions. It was concluded that the horsesof the police squad assembled Aracaju, are able to carry out patrol activities mounted, and that the stress testassociated with measurement of serum levels of CK, AST and LDH are important tools for determining the fitness.

Objetivou-se avaliar a performance e o limiar aeróbico de equinos da Cavalaria do Esquadrão de Polícia Montada do Estado de Sergipe, que atuam em atividades de baixa a média intensidade. Quinze equinos, mestiços de idades entre oito e 18 anos, foram submetidos ao teste de esforço a campo, com duração 20 minutos divididos em seis velocidades (de 2,5 a 9 rn/s), Durante e pós-teste foram coletadas amostras para a mensuração de lactato desidrogenase (LDH) (repouso e velocidades de O, 5,0 9,0 2,5 m/s e pós 30 minutos), creatina quinase (CK) easpartato aminotransferase (AST) (repouso e pós 30 minutos, 12 e 14 horas). Previa e posteriormente ao teste de esforço foram realizados exames de endoscopia respiratória. Ao exame endoscópio, pré-teste de esforço, foi observado a presença de alterações no sistema respiratório em 46,66% (7/15) dos equinos havendo dois casos dehemiplegia laringeana e um de deslocamento dorsal de palato mole, alteração etmoidal, neoplasia epiglótica, neoplasiaosteofaringeana e secreção brônquica. Concluiu-se que os equinos do esquadrão de polícia montada de Aracaju,estão aptos a realização de atividades de patrulha montada, e que o teste de esforço associado à mensuração de valores sé ricos das enzimas CK, AST e LDH são importantes ferramentas para a determinação do condicionamento físico.

Avaliação física de equinos da cavalaria do estado de Sergipe / Equine physical evaluation cavalry Sergipe state / Evaliacion física equina estado caballería Sergipe

Este estudio tuvo como meta evaluar los caballos de competición y de umbral aeróbico Montada Escuadrón de Caballería de Ia Policía dei Estado de Sergipe, que trabajan en Ias actividades de baja intensidad media. Quince caballos, mestizas edades de ocho a dieciocho anos, fueron sometidos a Ia prueba de esfuerzo decampo, con una duración de 20 minutos divididos en seis velocidades (de 2,5 a 9 rn/s). Se recogieron durante y después de Ia prueba Ias muestras para Ia medición de lactato deshidrogenasa (LDH) (reposo y una velocidad de O,5,09,02,5 m 1 s después de 30 minutos), Ia creatina quinasa (CK) y aspartato aminotransferasa (AST) (reposo ydespués de 30 minutos, 12 y 14 horas). Previa y después se realizaron los exámenes de Ias pruebas de resistenciade broncoscopia. En endoscopio examen, esfuerzo previa a Ia prueba, se observó Ia presencia de cambios en elsistema respiratorio en eI46,66% (7/15) de los caballos hay dos casos de hemiplejía laríngea y Dorsal desplazamientodei paladar blando, el cambio etmoidal, el cáncer de Ia epiglotis, Ia neoplasia osteo faringeana y Ias secreciones bronquiales. Se concluyó que los caballos de Ia patrulla de Ia policía montan Aracaju, son capaces de lIevar a caboactividades de patrulla montada, por 10 que Ia prueba de estrés asociado con Ia medición de los niveles sé ricos deCK, AST Y LDH son herramientas importantes para Ia determinación dei condicionamiento físico.(AU)

This study aimed to evaluate the performance and aerobic threshold horses Mounted Police Squadronof Cavalry of the State of Sergipe, who work in the average intensity low activities. Fifteen horses, crossbred ageseight to eighteen, were subjected to the stress test field, lasting 20 minutes divided into six speeds (from 2,5 to 9 mls). During and post-test samples were collected for measurement of lactate dehydrogenase (LDH) (rest and speedsof 0,5,0,9,0,2,5 m/s after 30 minutes), creatine kinase (CK) and aspartate aminotransferase (AST) (rest and after 30minutes, 12 and 14 hours). Preview and then the stress test bronchoscopy examinations were performed. Onexamination endoscope, pre-test effort, it was observed the presence of changes in the respiratory system in 46.66%(7/15) of horses there two cases of laryngeal hemiplegia and one dorsal displacement of the soft palate, ethmoidchange, epiglottis neoplasia, osteofaringeana neoplasia and bronchial secretions. It was concluded that the horsesof the police squad assembled Aracaju, are able to carry out patrol activities mounted, and that the stress testassociated with measurement of serum levels of CK, AST and LDH are important tools for determining the fitness.(AU)

Objetivou-se avaliar a performance e o limiar aeróbico de equinos da Cavalaria do Esquadrão de Polícia Montada do Estado de Sergipe, que atuam em atividades de baixa a média intensidade. Quinze equinos, mestiços de idades entre oito e 18 anos, foram submetidos ao teste de esforço a campo, com duração 20 minutos divididos em seis velocidades (de 2,5 a 9 rn/s), Durante e pós-teste foram coletadas amostras para a mensuração de lactato desidrogenase (LDH) (repouso e velocidades de O, 5,0 9,0 2,5 m/s e pós 30 minutos), creatina quinase (CK) easpartato aminotransferase (AST) (repouso e pós 30 minutos, 12 e 14 horas). Previa e posteriormente ao teste de esforço foram realizados exames de endoscopia respiratória. Ao exame endoscópio, pré-teste de esforço, foi observado a presença de alterações no sistema respiratório em 46,66% (7/15) dos equinos havendo dois casos dehemiplegia laringeana e um de deslocamento dorsal de palato mole, alteração etmoidal, neoplasia epiglótica, neoplasiaosteofaringeana e secreção brônquica. Concluiu-se que os equinos do esquadrão de polícia montada de Aracaju,estão aptos a realização de atividades de patrulha montada, e que o teste de esforço associado à mensuração de valores sé ricos das enzimas CK, AST e LDH são importantes ferramentas para a determinação do condicionamento físico.(AU)

Efeito da exposição aos crioprotetores glicerol e metilformamida sobre a viabilidade e fertilidade do sêmen equino

O processo de congelação e descongelação de sêmen induz a diversos efeitos deletérios na célula espermática. Crioprotetores são adicionados aos meios de congelação para minimizar esses danos, entretanto altas concentrações dessas substâncias aumentam sua toxicidade e, conseqüentemente, diminui a fertilidade. É importante o equilíbrio entre habilidade crioprotetora e toxicidade desses componentes. As amidas têm se mostrado um crioprotetor de baixa toxicidade, especialmente para garanhões de má congelabilidade e para outras espécies de animais. O presente estudo objetivou determinar a viabilidade e o efeito contraceptivo dos crioprotetores glicerol (GL) e metilformamida (MF) para congelação de sêmen de garanhões. EXPERIMENTO I: Foi colhido o sêmen de 10 garanhões com uma vagina artificial, diluído (1:1) em meio à base de leite (Botu-sêmen®) e centrifugado (600xg/10min). Foram ressuspendidos os pellets com meio de congelação (INRA82) contendo três concentrações diferentes de GL (2, 3 e 4,5%) e MF (2, 3 e 4,5%) e um grupo controle (Contr.) sem adição de crioprotetor, na concentração de 100x106 espermatozóides/mL. O sêmen foi envasado em palhetas de 0,5mL, resfriado a 5°C/1hora e mantido em vapor de nitrogênio durante 15 minutos e daí imerso em nitrogênio líquido. As amostras foram avaliadas após o período de estabilização (5°C/1hora) bem como após a descongelação (46°C/20seg) pelo sistema computadorizado (HTMA-IVOS-10). Foram avaliados parâmetros de motilidade, e através de Microscopia de Fluorescência, foi avaliada a integridade de membranas (plasmática e acrossomal) e potencial da membrana mitocondrial. A análise estatística foi realizada por ANOVA e teste de Tukey com significância de 5%. No período pós-estabilização a única diferença observada foi no parâmetro de motilidade progressiva, onde o grupo MF2% (25,7%) foi superior...

The frozen thawing semen process induce several damages on sperm cells. The use of cryoprotectors is fundamental to protect the spermatozoa from frozen-thawing damage, however high concentrations of these compounds can decrease the fertility. It is very important to balance the ability to cryoprotect with cryoprotector toxicity. Amidas has been shown to have a low toxicity specially for bad freezer stallions and some other species. The present experiment aimed to determine the effect of exposition on glycerol (GLY) and methylformamida (MF) over viability and fertility of stallion semen. On experiment I one ejaculated from 10 stallions was collected and diluted (1:1) in skim milk extender (Botu-sêmen®) and centrifuged (600g/10min). The sperm pellets were resuspended with a frozen extender (INRA82) containing 3 different concentrations of GLY (2, 3 or 4,5%) and MF (2, 3 or 4,5%) and a control group. (Contr.) using a concentration of de 100x106 esperm/mL. Semen was packaged in 0,5mL straws, cooled at a 5°C/1hour, maintained over nitrogen and finally frozen. The motility parameters were evaluated after stabilization (5°C/1hour) and again after frozen thawing (46°C/20seg) by CASA (HTMA-IVOS-10). Membrane integrity (Plasmatic and Acrossomal) and mitochondrial potential of membrane were also evaluated using fluorescent probes. ANOVA and Tukey test were used for statistical evaluation. After the stabilization time the only parameter that was different was the progressive motility (PM) that was higher on MF2% group (25,7%) when compared with Control Group (13,5%). No differences (p>0.05) were observed on Total Motility (TM) when concentrations of the same cryoprotector were compared, however differences favorable to MF were detected when comparisons were