Meios de preservação de sêmen ovino à base de água de coco em pó e extrato liofilizado de Aloe vera em protocolo de refrigeração a 4°C por 48 horas / Preservation medium based of lyophilized extract of Aloe vera and powdered coconut water (ACP 102®) refrigerated ram semen at 4°C for 48h

tThe additives of animal origin including egg yolk, widely used for sperm preservation. In order tominimize the impact on the use of animal products, the objective of this study was to evaluate the effect oflyophilized extract of Aloe vera and ACP 102® in ram semen refrigerated at 4°C for 48h. Ejaculates from threeram were split into three aliquots and diluted in ACP®, ACP®/ALV and ALV. Motility and viability was evaluatedafter sperm dilution, 2h, 24h and 48h of storage at 4°C. Results showed that there was a reduction in total andprogressive motility of fresh as compared to other storage time. No differences were observed in viability of freshand 2h as well as there was no difference of semen at for 24h and 48h storage. In conclusion, study showed that theaddition of lyophilized Aloe vera extract was able to maintain semen viability similar to ACP.(AU)

Meios de preservação de sêmen ovino à base de água de coco em pó e extrato liofilizado de Aloe vera em protocolo de refrigeração a 4°C por 48 horas / Preservation medium based of lyophilized extract of Aloe vera and powdered coconut water (ACP 102®) refrigerated ram semen at 4°C for 48h

tThe additives of animal origin including egg yolk, widely used for sperm preservation. In order tominimize the impact on the use of animal products, the objective of this study was to evaluate the effect oflyophilized extract of Aloe vera and ACP 102® in ram semen refrigerated at 4°C for 48h. Ejaculates from threeram were split into three aliquots and diluted in ACP®, ACP®/ALV and ALV. Motility and viability was evaluatedafter sperm dilution, 2h, 24h and 48h of storage at 4°C. Results showed that there was a reduction in total andprogressive motility of fresh as compared to other storage time. No differences were observed in viability of freshand 2h as well as there was no difference of semen at for 24h and 48h storage. In conclusion, study showed that theaddition of lyophilized Aloe vera extract was able to maintain semen viability similar to ACP.

EFEITO DE MEIOS DILUIDORES NA CONGELABILIDADE DO SÊMEN CANINO

O estudo tem por objetivo avaliar o efeito de dois meios diluentes para a congelação de espermatozoides caninos. Para tanto, foram utilizados três cães machos, adultos, de diferentes raças, com idades entre 2 a 5 anos e fertilidade comprovada. Foram realizadas quatro colheitas de sêmen de cada animal. As amostras colhidas foram divididas em dois grupos, grupo TRIS e o grupo BOTUDOG, com concentração de 80 x 106 espermatozoides por mL. Foram avaliados os parâmetros de cinética espermática e integridade de membrana plasmática e acrossomal (MPAI, %). Para as análises estatísticas foram realizadas pelo programa Statistical Analyses System. Verificou-se que os parâmetros de motilidade total (%), motilidade progressiva (%), velocidade linear progressiva (VSL; m/s), velocidade média da trajetória (VAP; m/s), linearidade (%), percentagem de espermatozoides rápidos (%) e integridade de membrana plasmática e acrossomal avaliados por citometria de fluxo foram superiores no Grupo 2, em que as amostras foram diluídas em meio comercial Botudog®, previamente a congelação. Estes dados demonstram que, o protocolo de congelação de sêmen canino, utilizando o diluente Botudog® mantém os parâmetros de cinética e viabilidade espermática, quando comparados ao meio TRIS gema.

The objective of this study was to evaluate the effect of two diluent media for the freezing of canine spermatozoa. For this, three adult male dogs of different races, aged between 2 to 5 years and proven fertility were used. Four semen samples were collected from each animal. The samples collected were divided into two groups, TRIS group and BOTUDOG group, with a concentration of 80 x 106 spermatozoa per mL. The parameters of spermatic kinetics and plasma and acrosomal membrane integrity (MPAI,%) were evaluated. Statistical analyzes were performed by the Statistical Analyzes System. The parameters of total motility (%), progressive motility (%), linear linear velocity (VSL; m / s), mean trajectory velocity (VAP; m / s), linearity (%), percentage of spermatozoa (%) and plasma membrane and acrosomal integrity assessed by flow cytometry were higher in Group 2, where the samples were diluted in Botudog® commercial medium, prior to freezing. These data demonstrate that the canine semen freezing protocol using the Botudog® diluent maintains the parameters of kinetics and sperm viability when compared to the TRIS gem medium.

Efeitos do trimetilaminoetano (TES) e ringer lactato em sêmen de macacos-aranha mantidos em cativeiro ( Ateles paniscus e A. marginatus) / Effects of TES and a ringer-lactato diluent in semen from captive spider monkeys (Ateles paniscus e A. marginatus)

The performances of the diluents TES and CEBRAN II were compared as cryopreservatives of semen from non human primates of the genus Ateles. The experiment was carried out using one Ateles marginatus and two Ateles paniscus specimens, males and adults, maintained in the same captivity conditions at the National Center of Primates (CENP-SVS/MS). The animals were subjected to clinical and andrological examinations - testicular biometry - before the semen collection by eletroejaculation. Evaluations of motility and forward movement in the fresh semen were made. Semen were made dilution was made with the diluents TES and CEBRAN II. The ejaculates were diluted with the diluents (2:1proportion), packed in 0.25mL plastic straws and cryopreserved in liquid nitrogen. After thawing, the packed ejaculates were appraised in thermo resistance test (TTR). The averages of volume and concentration were, respectively, 1.94mL (0.83) and 3,020,000 sptz/mL (275.97). The pH 8 and seminal coagulation were observed in all samples. The results suggest that the TES diluent presents better efficiency in the preservation of Ateles semen than CEBRAN II.(AU)

Assessing the growth and recovery of Salmonella Enteritidis SE86 after sodium dichloroisocyanurate exposure

The objective of the present study was to assess the growth and the recovery of Salmonella (S.) Enteritidis SE86 in different diluents, culture media and using different plating methods after the exposure to 200 mg/kg sodium dichloroisocyanurate (NaDCC). Before and after NaDCC exposure, SE86 was cultured at 30 °C and 7 °C in the following diluents: Peptone water (P), Saline solution (SaS), Peptone water+Saline solution (P+SaS), Peptone water+Tween 80+Lecithin+Sodium thiosulfate (P+N) and Saline solution+Tween 80+Lecithin+Sodium thiosulfate (SaS+N). The SaS diluent was chosen because it was able to maintain cells viable without growth and was further used for plating SE86 on non selective medium (Tryptic Soy Agar-TSA) and on selective media (Mannitol Lysine Crystal Violet Brilliant Green Agar-MLCB, Brilliant Green Agar-BGA, Salmonella Shigella Agar-SS and Xylose Lysine Dextrose-XLD). The Thin Agar Layer method (TAL) i.e., selective media overlayed with non selective TSA was also evaluated. Results indicated that SE86 not exposed to NaDCC was able to grow in P, P+N, SaS+N and P+SaS, but not in SaS, that was able to maintain cells viable. SE86 exposed to NaDCC demonstrated similar counts after dilution in SaS and the plating on non selective TSA, selective media MLCB, BGA, SS and XLD and on TAL media. SE86, S. Typhimurium and S. Bredeney, exposed or not exposed to NaDCC, showed no significant differences in counts on TSA, XLD and XLD overlayed with TSA, suggesting that all those media may be used to quantify NaDCC-exposed Salmonella by plating method.(AU)

Produção e preservação do sêmen de galos / Production and preservation of rooster semen

A seleção de reprodutores capazes de transmitir características de valor econômico para a progênie é fundamental para o sucesso da indústria de aves. Nesse sentido, os machos devem produzir sêmen de boa qualidade, com volume e concentração adequados. O ejaculado de galos é composto por uma grande quantidade de células espermáticas suspensas em um pequeno volume de plasma seminal, proveniente dos túbulos seminíferos. Por esse motivo, a adição de diluentes ao sêmen é uma prática de rotina nos estabelecimentos que realizam inseminação artificial, com a finalidade de aumentar o aproveitamento do ejaculado. A diluição também permite manter a qualidade do sêmen por várias horas, quando resfriado, ou por períodos mais longos, quando congelado. Para a manutenção da capacidade fertilizante do sêmen resfriado, substratos energéticos e agentes tampões devem ser incluídos no diluente. Para diminuir os danos sofridos pelos espermatozoides durante o congelamento, crioprotetores são adicionados ao sêmen. Entre eles, a dimetilacetamida vem sendo o mais utilizado nos protocolos para o congelamento de sêmen de galos nos últimos anos. A modulação lipídica do espermatozoide, por meio de dietas ou por adição de gema de ovo ou de seus derivados ao diluente, também auxilia na preservação do sêmen. Os benefícios são ainda maiores quando antioxidantes são incluídos.(AU)

Selecting breeders capable of transmit characteristics of economic value to the offspring is fundamental to poultry industry success. In this sense, males must produce good quality semen, with appropriate volume and concentration. Rooster ejaculates are composed of a great amount of sperm cells suspended in a small volume of seminal plasma, originated in the seminiferous tubules. For this reason, adding diluents to semen is a common practice in artificial insemination establishments, aiming to increase ejaculate use. Dilution also allows semen quality maintenance for hours, when refrigerated, or for longer periods, when frozen. To keep fertilizing capacity of refrigerated sperm, energetic substrates and buffer agents must be included into the diluents. To decrease sperm damage during freezing, cryoprotectants are added to the semen. Among them, dimethylacetamide has been the most used in freezing protocols for rooster semen in the last years. Sperm lipid modulation, by dietary means or by adding egg yolk or its derivates to the diluents, also helps in semen preservation. Benefits are improved when antioxidants are included.(AU)

Produção e preservação do sêmen de galos / Production and preservation of rooster semen

A seleção de reprodutores capazes de transmitir características de valor econômico para a progênie é fundamental para o sucesso da indústria de aves. Nesse sentido, os machos devem produzir sêmen de boa qualidade, com volume e concentração adequados. O ejaculado de galos é composto por uma grande quantidade de células espermáticas suspensas em um pequeno volume de plasma seminal, proveniente dos túbulos seminíferos. Por esse motivo, a adição de diluentes ao sêmen é uma prática de rotina nos estabelecimentos que realizam inseminação artificial, com a finalidade de aumentar o aproveitamento do ejaculado. A diluição também permite manter a qualidade do sêmen por várias horas, quando resfriado, ou por períodos mais longos, quando congelado. Para a manutenção da capacidade fertilizante do sêmen resfriado, substratos energéticos e agentes tampões devem ser incluídos no diluente. Para diminuir os danos sofridos pelos espermatozoides durante o congelamento, crioprotetores são adicionados ao sêmen. Entre eles, a dimetilacetamida vem sendo o mais utilizado nos protocolos para o congelamento de sêmen de galos nos últimos anos. A modulação lipídica do espermatozoide, por meio de dietas ou por adição de gema de ovo ou de seus derivados ao diluente, também auxilia na preservação do sêmen. Os benefícios são ainda maiores quando antioxidantes são incluídos.

Selecting breeders capable of transmit characteristics of economic value to the offspring is fundamental to poultry industry success. In this sense, males must produce good quality semen, with appropriate volume and concentration. Rooster ejaculates are composed of a great amount of sperm cells suspended in a small volume of seminal plasma, originated in the seminiferous tubules. For this reason, adding diluents to semen is a common practice in artificial insemination establishments, aiming to increase ejaculate use. Dilution also allows semen quality maintenance for hours, when refrigerated, or for longer periods, when frozen. To keep fertilizing capacity of refrigerated sperm, energetic substrates and buffer agents must be included into the diluents. To decrease sperm damage during freezing, cryoprotectants are added to the semen. Among them, dimethylacetamide has been the most used in freezing protocols for rooster semen in the last years. Sperm lipid modulation, by dietary means or by adding egg yolk or its derivates to the diluents, also helps in semen preservation. Benefits are improved when antioxidants are included.

Cryopreservation of Equine Semen Loaded in Cryovials

Background: Over the past twenty years the assisted reproduction techniques reached a rapid advance in domestic species of economic interest. By the time the male gamete has provided to be the most successful tool used to improve animal breeding programs. Freezing and stock of semen is a safe procedure to preserve reproductive potential of animals with superior genetic heritage. In the horse industry, unlike observed in ruminants, the development of sperm cryopreservation techniques is very slow but despite the technical barriers the artificial insemination with frozen semen is growing. Materials, Methods & Results: This experiment was divided in three steps. The first one compared the use of different diluents and freezing techniques. The other two stages were the use of cryogenic tubes for storing semen and the determination of mare pregnancy rates that were artificially inseminated with frozen semen packaged in cryogenic tubes. Six stallions were submitted to semen collection and twelve mares were artificially inseminated at first estrus of the breeding season. The samples were diluted in INRA82 or Nagase, containing 5% glycerol, with a concentration of 200 x 10 6 sperm/mL. A fraction of the diluted semen was stored in straws (0.5 mL), some of those straws were immediately frozen. The remaining straws were cooled from room temperature (± 24°C) to 5°C before freezing. The samples were thawed in a water bath at 37°C during 30 s. The same protocol with cooling prior to freezing was performed with samples filled into cryogenic tubes, and these samples were thawed in a water bath at 50°C during 100 s. The in vivo efficiency of cryopreserved semen was determined through artificial insemination (AI). The pregnancy diagnosis was performed after 20 days by ultrasound examination. After analyzing the data it was found that there was no significant difference in sperm motility and vigor among the tested diluents. [...]

Cryopreservation of Equine Semen Loaded in Cryovials

Background: Over the past twenty years the assisted reproduction techniques reached a rapid advance in domestic species of economic interest. By the time the male gamete has provided to be the most successful tool used to improve animal breeding programs. Freezing and stock of semen is a safe procedure to preserve reproductive potential of animals with superior genetic heritage. In the horse industry, unlike observed in ruminants, the development of sperm cryopreservation techniques is very slow but despite the technical barriers the artificial insemination with frozen semen is growing. Materials, Methods & Results: This experiment was divided in three steps. The first one compared the use of different diluents and freezing techniques. The other two stages were the use of cryogenic tubes for storing semen and the determination of mare pregnancy rates that were artificially inseminated with frozen semen packaged in cryogenic tubes. Six stallions were submitted to semen collection and twelve mares were artificially inseminated at first estrus of the breeding season. The samples were diluted in INRA82 or Nagase, containing 5% glycerol, with a concentration of 200 x 10 6 sperm/mL. A fraction of the diluted semen was stored in straws (0.5 mL), some of those straws were immediately frozen. The remaining straws were cooled from room temperature (± 24°C) to 5°C before freezing. The samples were thawed in a water bath at 37°C during 30 s. The same protocol with cooling prior to freezing was performed with samples filled into cryogenic tubes, and these samples were thawed in a water bath at 50°C during 100 s. The in vivo efficiency of cryopreserved semen was determined through artificial insemination (AI). The pregnancy diagnosis was performed after 20 days by ultrasound examination. After analyzing the data it was found that there was no significant difference in sperm motility and vigor among the tested diluents. [...](AU)

Comparison of five extenders for canine semen freezing / Comparação entre cinco diluidores na congelação de sêmen de cães

Semen was collected from six stud dogs to compare five extenders in the semen freezing process. Each ejaculate was divided in five parts and added to tris-fructose-citric acid, glicine, lactose, skim milk and tris-fructose-citrate extenders. The semen was diluted at 37C in extenders without glycerol, in the ratio 1:1 and cooled for 60 minutes to reach a 5C temperature. Then, extenders with glycerol in the ratio of 2:1 were added to give the final prefreezing concentration of 4% of glycerol. The diluted semen with the cryoprotectant was maintained for a further 60 minutes in refrigeration to equilibrate the spermatozoa in the glycerol and packaged in 0.5 ml plastic straws. The straws were maintained for 30 minutes in vapor, plunged and stored in liquid nitrogen. Sperm morphology was evaluated before and after freezing, whereas progressive motility (%) and velocity of forward progression (0-5) were appraised in different periods of the freezing process. The extender tris-fructose-citric acid showed the best post thaw progressive motility and velocity of forward progression compared to the others extenders. Semen freezing increased major sperm morphological abnormalities, regardless of the extender.

Foram utilizados ejaculados de 6 cães para comparar cinco diluidores no processo de congelação de sêmen. Cada ejaculado foi dividido em 5 partes e adicionadas aos diluidores tris-fructose-ácido cítrico, glicina, lactose, leite desnatado e tris-fructose-ácido cítrico. O sêmen foi diluído a 37C sem adição de glicerol na proporção 1:1 (fração A) e refrigerado durante 60 minutos até atingir a temperatura de 5ºC, quando foi adicionada a fração dos diluidores contendo glicerol (fração B) na proporção 2:1, atingindo a concentração final de 4% de glicerol. O sêmen diluído permaneceu por 60 minutos em refrigeração para equilíbrio no glicerol, sendo envasado em palhetas de 0,5 ml, mantido por 30 minutos no vapor de nitrogênio e imerso e armazenado em nitrogênio líquido. Foram avaliados a motilidade progressiva retilínea, o vigor e os defeitos espermáticos antes da congelação e após a descongelação do sêmen em água a 37C. Os resultados mostraram que os diluidores tris-fructose-ácido cítrico e glicina apresentaram as melhores médias de motilidade progressiva retilínea e de vigor espermático após a descongelação. A congelação aumentou a freqüência dos defeitos espermáticos maiores independente do diluidor.

Pré-diluição e congelação de sêmen suíno em água de coco in natura, após três diferentes pré-tratamentos de incubação / Boar semen pre-diluted or freezing with coconut water in natura after three different pre-treatments

Foram utilizados vinte e quatro ejaculados de cinco diferentes cachaços na congelação de sêmen suíno, sendo doze deles pré-diluídos em água de coco in natura e em Merck I, utilizando a lactose como diluidor de refrigeração e de congelação (experimento A) e doze pré-diluídos apenas com Merck I. Após três diferentes pré-tratamentos, a água de coco in natura foi utilizada como diluidor de refrigeração e de congelação, sendo a lactose utilizada como controle (experimento B). Seguiu-se a metodologia convencional de congelação de sêmen desta espécie -Tierãrztliche Hochschule Hannover (grupo 1A-1B) e um novo processo com longo período de equilíbrio (grupos 2A-2B e 3A-3B). A qualidade do sêmen descongelado foi avaliada pela motilidade subjetiva (SMOT), motilidade computadorizada (CMOT) e morfologia das células com borda apical normal (NAR), após fixação em formol citrato. As porcentagens de espermatozóides com NAR foram 65 por cento (grupo 1A), 71 por cento (grupo 2A) e 75 por cento (grupo 3A) para o sêmen pré-diluído em água de coco e 60 por cento (grupo 1A), 68 por cento (grupo 2A) e 68 por cento (grupo 3A) para aquele pré-diluído com Merck I (experimento A); e 56 por cento (grupo 1B), 68 por cento (grupo 2B) e 73 por cento (grupo 3B) para o sêmen congelado em água de coco e 60 por cento (grupo 1B), 68 por cento (grupo 2B e 3B) para o sêmen congelado em lactose (experimento B). Não havendo, portanto, diferença estatística entre os dois pré-diluentes e os dois diluentes de refrigeração e de congelação (p<0,05). Concluiu-se, portanto, que 1) os pré-tratamentos com longo período de equilíbrio têm melhor efeito na proteção do acrossoma do espermatozóide suíno e para manter a motilidade espermática e 2) a água de coco como pré-diluente e diluente para refrigeração e de congelação é semelhante ao Merck I (experimento A) e a lactose (experimento B), sendo portanto indicado para pré-diluir e congelar sêmen suíno. (AU)

Twenty-four ejaculates were collected from five boars for the conventional freezing procedure at the Veterinary School of Hannover (group 1A-1B) and for an extended holding time procedure (group 2A-2B and 3A-3B). The semen were prediluted in two different diluents, coconut water in natura and Merck I medium before freezing (experiment A) and prediluted only with Merck I, but cooling and freezing with coconut water in natura or lactose (experiment B). The semen were arranged into three groups according to the different holding periods before freezing, group 1A-1B (sperm-rich phase, preserved for 4 hours by 15°C), group 2A-2B (sperm-rich phase, preserved for 16 hours by 18°C) and group 3A-3B (total semen, preserved for 16 hours by 18ºC). The quality of the thawed boar spermatozoa was evaluated by subjective motility (SMOT), computerassisted motility (Cell Motion Analyser, Strömberg-Mica CMOT) and morphology of acrosomal ridges (NAR) after fixation in formol citrate. The acrosomal integrity (NAR) was 65% (group 1A), 71% (group 2A) and 75% (group 3A) for semen prediluted with coconut water and 60% (group 1A), 68% (group 2A) and 68% (group 3A) for semen prediluted with Merck I medium, respectively (experiment A); and 56% (group 1B), 68% (group 2B) and 73% (group 3B) for semen freezing with coconut water diluent, and 60% group 1B), 68% (group 2B and 3B) for semen freezing with lactose (experiment B). Thus, did not differ significantly (p<0.05) between both pre-diluents and both freezing diluents. However, the post thaw motility by both SMOT and CMOT and the acrosome integrity (NAR) were significantly higher (p<0.05) in the semen preserved for extended holding time (group 2A-2B and 3A-3B) than those for short time (group 1A-1B). It is concluded that the extended holding time has a better effect to protect acrosome of boar spermatozoa and also maintain sperm motility. Therefore, preserving boar semen for longer period before freezing is indicated for subsequent research on in vitro research with boar semen. And because the results using coconut prediluent and diluent were similar to that using Merck I medium (experiment A) and using lactose (experiment B), it is also indicated to use coconut for diluent and for freezing boar semen.(AU)