A pirapitinga (Piaractus brachypomus) Ã uma espÃcie de peixe exÃtico introduzido no nordeste brasileiro o qual tem grande importÃncia comercial, devido a seu hÃbito alimentar, excelente crescimento, resistÃncia ao manejo em cativeiro e Ãs enfermidades. A criopreservaÃÃo de seu sÃmen pode contribuir nos aspectos econÃmico, genÃtico e meio ambiental da regiÃo e da espÃcie. O objetivo deste trabalho foi desenvolver um protocolo adequado de congelaÃÃo seminal para poder obter motilidades prÃximas ao sÃmen a fresco. Trabalhou-se com o sÃmen d e19 machos maduros de dezembro de 2007 a marÃo de 2008, determinando as caracterÃsticas seminais; a toxicidade dos crioprotetores DMSO 10% e metilglicol 10%, diluÃdos em glicose 5%, ACP 104 e soluÃÃo Ringer das diluiÃÃes de 1:3, 1:5 e 1:7 durante 15 minutos a 4Â C; a criopreservaÃÃo a -196Â C com as mesmas condiluiÃÃes usando palhetas de 0,25 e 0,5 mL, congeladas em vapores de nitrogÃnio lÃquido em caixas de isopor ou em âdry shipperâ, sendo depois transferidas para botijÃes criogÃnicos durante 15 dias. Foram avaliadas as motilidades e velocidades atravÃs da anÃlise seminal auxiliado por computador (CASA) pÃs-descongelaÃÃo; e a morfologia seminal fresco e pÃs-descongelaÃÃo. Os parÃmetros seminais a fresco observados foram: motilidade objetiva mÃdia de 94%, concentraÃÃo de 94 x 10 spzt/mL, volume d e7 mL, Osmolaridade de 317 mOsm/Kg e ph de 8,4. Nos testes de toxicidade nÃo foram encontradas diferenÃas significativas entre o T3 na diluiÃÃo 1:3 comparado com o grupo controle (P=0.06), apresentando motilidade de 80% e VCL de 95 um/s. O melhor mÃtodo de congelamento foi o âdry shipperâ utlizando palhetas de 0,5 mL registrando motilidade de 625 para o T6 na diluiÃÃo de 1:7, entretando, apresentaram baixas velocidades para todos os tratamentos, com melhores resultados de VCL, VSL e VAP para o T6. No sÃmen fresco a morfoanomalia mais encontrada foi a cauda dobrada (20%) e no sÃmen pÃs-descongelaÃÃo, a cauda enrolada (23%). Comparando-se os resultados de morfoanomalia antes e deposi do pÃs-descongelamento foram observadas diferenÃas significativas entre as amostras (P<0,05). Concluiu-se que o sÃmen da Piratininga pode ser preservado em Ringer+Retilglicol 105 em 1:3, sem apresentar altas toxicidades, e criopreservado em Glicose+DMSO 10% utilizando o âdry shipperâ. / The pirapitinga (Piaractus brachypomus) is a species of exotic fish introduced in northeastern Brazil which has great commercial importance due to their feeding habits, excellent growth, resistance management and diseases in captivity. The cryopreservation of their semen may contribute to the economic, environmental and genetic and species of the region. The objective of this study was to develop a protocol suitable for freezing sperm motility to get close to the fresh semen. Worked with the mature male sperm d e19 December 2007 to March 2008, determining the seminal characteristics, the toxicity of the cryoprotectants DMSO and 10% methylglycol 10%, diluted in glucose 5%, ACP 104 and the dilution of Ringer solution 1:3, 1:5 and 1:7 for 15 minutes at 4 Â C, the cryopreservation to -196 Â C with the same condiluiÃÃes using straws of 0.25 and 0.5 mL, frozen in liquid nitrogen vapors in styrofoam boxes or "dry shipper" and then transferred to cryogenic botijÃes for 15 days. We evaluated the motility and velocity through the computer-aided sperm analysis (CASA) after thawing, and sperm morphology fresh and post-thawing. The seminal parameters were observed in fresh: motility average objective of 94%, concentration of 94 x 10 spzt / mL, d e7 mL volume, osmolarity of 317 mOsm / kg and pH of 8.4. In toxicity tests were not significant differences between the T3 in 1:3 dilution compared to the control group (P = 0.06), showing 80% motility and 95 of a VCL / s. The best method of freezing was the "dry shipper" user straws of 0.5 mL recording motility of 625 for T6 in dilution of 1:7, entertain, showed low rates for all treatments, with best results of VCL, VSL and VAP for the T6. In the fresh semen was found more morfoanomalia the tail folded (20%) and the sperm after thawing, the coiled tail (23%). Comparing the results of morfoanomalia deposited before and after the thawing were significant differences between samples (P <0.05). It was concluded that the semen can be preserved in Piratininga Ringer + Retilglicol 105 in 1:3, without high toxicity, and cryopreserved in Glucose + 10% DMSO using the "dry shipper".

Se emplearon 32 muestras de semen procedentes de cuatro ovinos a fin de ser criopreservadas adicionándoles dos antioxidantes:Tempo (2,2,6,6 tetrametil-1-piperidiniloxil) y Tempol (4-hidroxi 2,2,6,6 tetrametil-1-piperidiniloxil), cada uno en concentración de 0.5, 1.0 y 2.5 mM. La finalidad del trabajo fue evaluar el efecto postdescongelamiento que pudiera tener sobre la motilidad progresiva, la viabilidad e integridad acrosomal, y la capacitación espermática prematura. Para cada concentración de cada antioxidante y para el grupo control, se utilizó un dilutor en base a Tris, realizándose 8 repeticiones, cada una con 4 muestras de semen. Los resultados obtenidos demuestran que la adición de Tempo a una concentración de 0.5 mM mejora significativamente la calidad del semen criopreservado en comparación con el grupo control, incrementado los porcentajes de motilidad progresiva (79 vs. 67 %), viabilidad e integridad acrosomal (70 vs. 58 %) y reduciendo la capacitación espermática prematura (9 vs. 15 %). Sin embargo, la adición de Tempol disminuyó la calidad seminal post descongelamiento en comparación con el control. Consecuentemente, el efecto de las especies reactivas de oxígeno sobre la calidad seminal puede ser reducido por la adición de Tempo 0.5 mM durante el enfriamiento. En conclusión la adición de Tempo 0.5 mM en un dilutor en base a Tris, al finalizar la fase de enfriamiento, podría constituir una estrategia para mejorar la calidad de semen ovino criopreservado y por lo tanto aumentar los porcentajes de fertilidad en la inseminación artificial en ovejas. / --- Trirty-two semen samples coming from 4 rams were frozed using 2 antioxidants, Tempo (2,2,6,6 tetramethyl-1-piperidinyloxyl) and Tempol (4-hidroxi 2,2,6,6 tetramethyl-1-piperidinyloxyl), each one in concentrations of 0.5, 1.0, and 2.5 mM, with the objective of evaluate their effects on post-thaw motility, viability, acrosomal integrity, and earlier sperm capacitation. It were realized 8 replication for each antioxidant concentrantion and control group, using 4 semen samples in each replication. Semen samples were diluted on a Tris extender. Results show that Tempo 0.5 mM improve frozen semen quality (P is less than 0.05) in comparison with control group, by increasing percentages of progresive motility (79 vs. 67 %), viability and acrosomal integrity (70 vs. 58 %), and decreasing earlier sperm capacitation (9 vs. 15 %). However, when Tempol was used, it was observed a decrease in frozed semen quality in comparison with control group. Consequently, the adverse effect of reactive oxygen species on semen quality could be reduced by the addition of Tempo 0.5 mM during the cooling process. In conclusion, the use of Tempo 0.5 mM on a Tris extender, at the end of the cooling process could be an alternative for improving the quality of frozed ram semen, and in this way, improve the fertility in artificial insemination in ewes. / Tesis

M.Sc. (Zoology) / This investigation is divided into two sections. Section A deals with aspects of the spermatology of the tiger fish and section B deals with the cryopreservation of spermatozoa of selected freshwater fish species.

Orientador: Fernandes Denardi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-07-24T05:44:48Z (GMT). No. of bitstreams: 1 Esteves_SandroCassiano_M.pdf: 4359044 bytes, checksum: ef86ac776f66f2a4dfc46a48740ef92b (MD5) Previous issue date: 1998 / Resumo: Apesar do aperfeiçoamento das técnicas de criopreservação, o potencial fértil do sêmen humano criopreservado ainda é insatisfatório. Por outro lado, o processamento do sêmen constitui etapa fundamental nas técnicas de reprodução assistida. A técnica de "swim-up" é o método mais freqüentemente utilizado para o processamento do sêmen humano, pois permite a seleção de um grande número de espermatozóides móveis. O objetivo do presente estudo foi avaliar se a seleção de espermatozóides humanos pela técnica de "swim-up", antes da criopreservação, é capaz de minimizar a perda de motilidade, vitalidade e integridade acrossômica, decorrentes do processo de congelamento-descongelamento. Visa-se, dessa forma, propiciar, após o descongelamento, a recuperação de espermatozóides com potencial fértil superior àqueles não-selecionados. Amostras de sêmen de 15 indivíduos com fertilidade comprovada foram obtidas por masturbação, após 2 a 3 dias de abstinência sexual, e divididas em duas alíquotas de volumes iguais. A primeira delas não recebeu qualquer tratamento (não¬selecionada), enquanto que a segunda foi processada pela técnica de "swim-up" (selecionada). Ambas as alíquotas foram, então, criopreservadas através da técnica de vapor de nitrogênio líquido. A motilidade percentual, e os parâmetros da motilidade espermática foram avaliados por um analisador computadorizado. A integridade acrossômica foi analisada pela técnica de fluorescência, empregando-se a lecitina do amendoim (FITC-PNA), em associação com o corte supravital bis-benzimida (Hoescht¬33258), para avaliação da vitalidade espermática. Cada alíquota foi avaliada quanto à motilidade percentual, parâmetros da motilidade espermática, vitalidade e integridade acrossômica, antes e depois da criopreservação. O teste "t" de Student para amostras pareadas, com nível de significância de 0,05, foi utilizado para determinar diferenças estatísticas na motilidade espermática, vitalidade e integridade acrossômica, bem como para avaliar as diferenças pecentuais destes parâmetros de antes para depois da criopreservação. Os espermatozóides selecionados pela técnica de "swim-up" apresentaram, antes da criopreservação, valores médios de vitalidade, motilidade percentual e parâmetros da motilidade espermática, com exceção da linearidade, significativamente superiores aos dos espermatozóides não-selecionados. Entretanto, não houve diferença estatística no percentual de espermatozóides com acrossomos intactos entre as amostras não-selecionadas e selecionadas. Após a criopreservação, observou-se, em ambas as amostras, queda significativa da motilidade percentual, parâmetros da motilidade espermática, vitalidade e integridade acrossômica. Contudo, não houve diferença estatística na variação percentual da motilidade e vitalidade espermática, de antes para depois da criopreservação, entre as amostras não-selecionadas e selecionadas pela técnica de "swim-up". Em contrapartida, a integridade acrossômica dos espermatozóides criopreservados foi significativamente superior nas amostras selecionadas por esta técnica. Além disso, a queda percentual da integridade acrossômica, depois da criopreservação, foi significativamente menor nas amostras selecionadas pela técnica de "swim-up". Embora a técnica de "swim-up" tenha selecionado a subpopulação de espermatozóides qualitativamente superior, antes da criopreservação, tal tratamento não foi eficaz para minimizar a perda de motilidade e vitalidade, decorrentes do processo de criopreservação. Entretanto, o processamento do sêmen pela técnica de "swim-up", anteriormente à criopreservação, permitiu a recuperação de um percentual maior de espermatozóides com acrossomos intactos, reduzindo a perda de integridade acrossômica, causada pelo processo de criopreservação. Portanto, a seleção de espermatozóides pela técnica de "swim-up", previamente à criopreservação, é benéfica para aumentar o potencial fértil dos espermatozóides criopreservados / Abstract: Despite of the developments in freezing techniques, the fertilizing potential of human spermatozoa after thawing remains poor. Sperm processing, on the other hand, is a fundamental step in assisted reproductive techniques. Swim-up is the most frequent used procedure for processing of human spermatozoa, as it selects a high number of motile spermatozoa. The aim of this study was to evaluate whether sperm selection by the swim¬up technique before freezing can minimize loss of motility, viability and acrosome integrity due to the freezing-thaw process, thus yielding post-thaw recovery of spermatozoa with higher fertilizing potential than unprocessed ones. Semen specimens from 15 healthy individuaIs with proven fertility were obtained by masturbation after 2 to 3 days of abstinence, and divided into two equal aliquots. The first aliquot received no treatment (unprocessed) and the second was processed by the swim-up technique (processed). Both aliquots were then cryopreserved by the liquid nitrogen vapor method. Percent motility and sperm motion characteristics in unprocessed and processed specimens were analyzed by computer-assisted semen analysis. Sperm viability was evaluated by a supra-vital dye bis-benzimide (Hoescht-33258). The acrosome integrity were assessed by fluorescein iso-thiocyanate conjugated peanut lectin (FITC-PNA). Each aliquot was evaluated for percent motility, motion characteristics, sperm viability and acrosome integrity either before freezing and afieI' thawing. The paired Student's "t" test was used to test for statistical differences in sperm motility, viability and acrosome integrity either before freezing and after thawing, as well as for percent change' on these parameters due to the cryopreservation processo A alpha leveI of 0.05 was considered significant. Compared with the unprocessed samples, the spermatozoa selected by the swim-up technique exhibited better percent motility, viability and all motion parameters, except linearity, before freezing. However, the frequency of spermatozoa exhibiting intact acrosomes was not different between unprocessed and swim-up specimens. After thawing, a significant impairment was observed in percent motility, motion characteristics, sperm viability and acrosome integrity in both unprocessed and swim-up specimens. Nevertheless, there was no difference in the percent change of sperm motility and viability from before freezing to after thawing between unprocessed and swim-up groups. However, acrosome integrity was significant1y higher in swim-up se1ected spermatozoa after thawing. In addition, the percent decrease in acrosome integrity from before freezing to after thawing was significant1y 10wer in swim-up se1ected specimens. Although sperm processing by the swim-up technique se1ected a better qua1ity sperm popu1ation before freezing, this treatment have not been ab1e to minimize the 10ss in moti1ity and viabi1ity due to the freeze-thaw processo However, pre-freeze se1ection of spermatozoa by the swim-up technique yie1ded a higher frequency of spermatozoa with intact acrosomes and reduced the 10ss in acrosome integrity due to the cryopreservation processo Therefore, pre-freeze sperm se1ection by swim-up may increase the ferti1izing potentia1 of spermatozoa after cryopreservation / Mestrado / Cirurgia / Mestre em Cirurgia

Determina la concentración óptima del glicerol, etilenglicol y dimetil sulfóxido en la criopreservación de espermatozoides epididimarios de alpaca. El análisis estadístico de los tres primeros experimentos se realiza mediante un modelo de regresión polinomial quíntuple, se obtiene la concentración óptima de 1.16, 1.02 y 0.9 M para el glicerol, etilenglicol y dimetil sulfóxido respectivamente. El cuarto experimento determina cuál de los crioprotectores criopreservaba mejor la motilidad, integridad de membrana plasmática y viabilidad e integridad acrosomal de los espermatozoides epididimarios. Está conformado por las concentraciones óptimas de los tres crioprotectores y en este se utiliza un análisis de varianza, además de la Prueba de Tukey. Concluye que las concentraciones de 0.9 M de DMSO y 1.16 M de glicerol conservan mejor las características espermáticas antes mencionadas a diferencia de la concentración de 1.02 M de etilenglicol. / Tesis

Espermatozoides criopreservados de Vicugna pacos “alpaca” fueron sometidos a condiciones estresantes que estarían asociadas con una sobreproducción de especies reactivas de oxigeno (ROS) y una disminución de su capacidad antioxidante lo que conlleva a la peroxidación lipídica de sus membranas reduciendo su viabilidad celular. El objetivo de este estudio fue investigar el efecto del suplemento α–Tocoferol en la movilidad, vitalidad, integridad funcional de membrana (HOST) e integridad acrosomal de espermatozoides de alpaca congelados/descongelados. Espermatozoides epididimarios obtenidos de 16 alpacas fueron criopreservados en medio TES-Tris-yema suplementado con distintas concentraciones de α–Tocoferol (0, 200, 500 y 1000 μg/mL usando el procedimiento de congelamiento lento en pajillas y almacenados a -196 ºC por un periodo mínimo de 30 días. Los resultados mostraron que los valores de motilidad espermática en los grupos suplementados fueron significativamente (p<0.05) mayores que el grupo no suplementado. El diluyente suplementado con 200 μg/mL de α–Tocoferol tuvo un valor de vitalidad significativamente más alto que con las otras concentraciones (p<0.05). Los porcentajes de HOST y acrosoma intacto fueron significativamente mejorados (p<0.05) por la suplementación con 200 μg/mL de α–Tocoferol comparado con el grupo control. En conclusión, α–Tocoferol, como suplemento, a una concentración de 200 μg/mL en el diluyente TES-Tris-yema durante la criopreservación tuvo un efecto positivo en la calidad espermática post-descongelamiento. / -- Cryopreserved sperm of Vicugna pacos “alpaca” were subjected to stressful conditions that would be associated with an overproduction of reactive oxygen species (ROS) and a decrease in antioxidant capacity which leads to lipid peroxidation of the membrane reducing cell viability. The aim of this study was to investigate the effect of α–Tocopherol supplementation on movility, viability, functional integrity of the membrane and acrosomal integrity of frozen/thawed alpaca sperm. Epididymal sperm collected from 16 alpacas were cryopreserved in TES-Tris-yolk medium supplemented with different concentrations of α–Tocopherol (0, 200, 500 and 1000 μg/mL, using the slow freezing procedure in straws and stored at -196 °C for a minimum period of 30 days. The results showed that the sperm motility values in the supplemented groups were significantly (p<0.05) higher than that unsupplemented group. The extender supplemented with 200μg/mL of α–Tocopherol had a vitality value significantly higher than that of other concentrations (p<0.05). The percentages of HOST and acrosome intact were significantly improved by supplementing with 200 μg/mL of α–Tocopherol compared with control group. In conclusion, α-Tocopherol, supplemented at 200 μg/mL concentration in TES-Tris-yema extender during cryopreservation had a positive effect on post-thawed sperm quality. / Tesis

El objetivo del presente trabajo fue evaluar el efecto del momento de adición del glicerol durante la curva de enfriamiento sobre la calidad espermática del semen canino. Para esto se utilizaron 15 eyaculados de 4 perros adultos. Se evaluó la motilidad, volumen, concentración espermática e integridad funcional de membrana de cada eyaculado. Cada muestra de semen fue sometida a 3 métodos de adición del glicerol (Tratamiento 1, Tratamiento 2 y Tratamiento 3) los cuales se diferenciaron por la concentración de glicerol presente en el dilutor durante la curva de enfriamiento. En el Tratamiento 1 el total del glicerol se adicionó al inicio de la curva de enfriamiento, en el Tratamiento 2 se adicionó una fracción del glicerol total al inicio de la curva de enfriamiento y la fracción restante al final de la curva, finalmente en el Tratamiento 3 el glicerol se añadió al final de la curva de enfriamiento. Finalizada la curva de enfriamiento el semen se envaso en pajillas de 0.5 mL para proceder al congelamiento en nitrógeno liquido. La motilidad progresiva y la integridad funcional de membrana fueron los parámetros utilizados para evaluar la calidad seminal al descongelamiento. No se encontró diferencia estadística entre ninguno de los 3 métodos de adición del glicerol, a pesar de esto el Tratamiento 1 mantenía ligeramente mejor la motilidad y la integridad funcional de membrana (44% y 46%) en comparación con el Tratamiento 2 (37% y 36%) y el Tratamiento 3 (38% y 37%). Esto nos demuestra que el momento de adición del glicerol no influye de manera diferencial sobre la motilidad progresiva y la integridad funcional de membrana pudiéndose gracias a esto simplificar el proceso de criopreservación al añadir el glicerol al inicio de la curva de enfriamiento pues ya no se manipularía el semen hasta el envasado. / --- The objetive of this experiment was to evaluate the effect of the moment of addition of the glycerol during the curve of cooling on the spermatic quality of the canine semen. For this there were used 15 ejaculated of 4 adult dogs. There was evaluated the motility, volume, spermatic concentration and functional integrity of membrane of every ejaculated. Every sample of semen was submitted to 3 methods of addition of the glycerol (Treatment 1, Treatment 2 and Treatment 3) which differentiated for the concentration of glicerol present in the dilutor during the curve of cooling. In the Treatment 1 the whole of the glycerol was added to the beginning of the curve of cooling, in the Treatment 2 added to himself a fraction of the entire glycerol to the beginning of the curve of cooling and the remaining fraction at the end of the curve, finally in the Treatment 3 the glycerol was added at the end of the curve of cooling. Finished the curve of cooling the semen was packed in straw hats of 0.5 ml to proceed to the freezing in liquid nitrogen. The progressive motility and the functional integrity of membrane were the parameters used to evaluate the seminal quality to the post-thawing. There was not statistical difference between any of 3 methods of addition of the glicerol, in spite of this the Treatment 1 was keeping lightly better the motility and the functional integrity of membrane (44 % and 46 %) compared to the Treatment 2 (37 % and 36 %) and the Treatment 3 (38 % and 37 %). This demonstrates us that the moment of addition of the glycerol does not influence in a distinguishing way the progressive motility and the functional integrity of membrane being able thanks to this to simplify the process of criopreservación after the glycerol to add to the beginning of the curve of cooling since the semen would not be already manipulated up to the stiff one. / Tesis

The diploma thesis: Study of Selected Physiological Features of Field Weed Populations from Different Habitats investigates the effects of temperature and habitat influences on seed germination of redroot pigweed Amaranthus retroflexus and German chamomile Matricaria chamomilla. Seeds of redroot pigweed were collected from the habitats: field margin, tree alley, railway and field. Seeds of chamomile were collected from the habitats: field margin, tree alley and were also bought. The seeds were exposed to temperatures 6 °C, 8 °C and 18 °C.The highest seed germination of both species was at 18 °C and the lowest was at 6 °C. The highest seed germination of redroot pigweed was recorded from the „field margin“ habitat 34,67 % and the lowest seed germination from the „field“ habitat 28,67 %. The highest seed germination of German chamomile was recorded from the „bought“ habitat 86,67 % and the lowest seed germination from the „field margin“ habitat 34 %.

A preliminary test was conducted evaluating the effect of digestible lysine (dLys) on broiler breeder (BB) male semen quality from forty-one to forty-nine wk of age. Five dietary treatments: corn-soybean meal diet with 1,000 mg dLys/rooser/day (Soy1,000), distillers dried grains with solubles diet with 1,000 (DDGS1,000), 850 (DDGS850), 700 (DDGS700), and 550 (DDGS550) mg dLys/rooster/day. Semen quality was similar except percentage dead sperm which was higher in DDGS550. A second trail was conducted using the same dietary treatments as the preliminary test during twenty to thirty-nine wk of age. Semen quality was similar except percentage dead sperm which was higher in Soy1,000. Soy1,000 exhibited higher body weight (BW), breast weight, and plasma testosterone. In conclusion dLys levels from 1,000 to 700 mg/rooster/day will not adversely affect semen quality of BB males. Attention should be given to BW in BB as it could lead to an increased percentage dead sperm.

The collection and preservation of llama semen is not a standardized procedure as it is in cattle. It has a number of complications that are mainly associated with the characteristics of the semen. In order to contribute to the knowledge of reproductive biology, we decided to evaluate the behavior of llama semen in relation to the different diluters in this study. This study was done at the National Germplasm Bank for Camelids which is part of the Ministry of Rural Development of Agriculture and Environment. The Ministry is located in the Agriculture Experiment Center of Condoriri which belongs to the College of Agriculture, Livestock, and Veterinary Sciences of the Technical University of Oruro. The University is located 49 km northeast of the province of Cercado in the department of Oruro and 12 km northeast of the community of Caracollo. The geographic coordinates of Caracollo are 17º31’41” south latitude and 67º14’02” west longitude. It is at an altitude of 3830 m above sea level and has an area of 1640 ha (Geographical map of the Military Geographical Institute). The macroscopic evaluations made were: pH (7.2 - 7.6), volume (2 - 4.5 cc), color (clear white or milky white), density, or consistency by simple inspection (subjective). The microscopic evaluations made were: motility (50 - 70%), viability (50 - 70%), concentration (20 - 40 x 107/esp./cc), and abnormalities (10% at most). All materials and environments used in this assessment were at room temperature. The analysis of variance test at 99% probability showed that the macroscopic and microscopic characteristics are affected by different harvest times. The two diluter preparations studied were 1) PBS diluter (with all its components that will serve the sperm as energy, nutrient, and cushioning), egg yolk, blood serum, and glycerol, and 2) TRIS diluter (with all its components that will serve the sperm as energy, nutrient, cushioning, and elements to avoid contamination), egg yolk, blood serum, and glycerol. All these materials were brought to 35 - 37ºC before being added to the semen. The prepared samples containing the diluent were then cooled to 5ºC. The samples were then collected for storage, with special care to maintain the sample temperature. The freezing was done slowly by placing the rack of containers at a given height of liquid nitrogen for seven to ten minutes. Following this, the containers were submerged. This slow freezing process prevented problems in the metabolism of the sperm after thawing. The samples were unthawed at intervals of 15 and 30 days by rapidly removing them from the liquid nitrogen and placing them in a water bath for 30 seconds. The evaluations of viability, motility, and concentration were all performed within 15 minutes of thawing. The results obtained from the analyses after thawing on average were better for the diluter PBS than for the diluter TRIS. The analysis of variance test at 99% probability showed that the characteristics after thawing were affected differently according to the diluter used in the dilution.