A arte de inseminar éguas com sêmen congelado / The art of inseminating mares with frozen semen

Nos últimos quinze anos, a equideocultura foi uma atividade em destaque com significativo crescimento no Brasil e no mundo. O Brasil é o segundo país no mundo que mais utiliza transporte de sêmen equino, ficando atrás apenas dos Estados Unidos e a utilização do sêmen congelado no país vem expandido a cada dia. O índice de prenhez por ciclo, com sêmen equino congelado é variável e oscila entre 25 e 40%. Adicionalmente, sabe-se que o sêmen de alguns garanhões apresenta baixa viabilidade após o descongelamento. A primeira prenhez obtida com sêmen equino congelado foi relatada há cinco décadas. Segundo Cazalez, 2020, é muito difícil recomendar uma dose inseminante padrão para sêmen congelado em equinos. A maioria dos estudos científicos não consegue controlar o efeito de outros fatores "de confusão" como método de processo, fator égua, fator garanhão, técnica de inseminação etc., tornando difícil uma comparação crítica dos mesmos. Rigby et al. (2001) obtiveram taxas de prenhez similares ao se comparar a inseminação artificial profunda em corno uterino com endoscópio e pipeta flexível.(AU)

In the last fifteen years, equine breeding has been a prominent activity with significant growth in Brazil and in the world. Brazil is the second country in the world that most uses equine semen transport, second only to the United States, and the use of frozen semen in the country is expanding every day. The pregnancy rate per cycle with frozen equine semen is variable and ranges from 25 to 40%. Additionally, it is known that the semen of some stallions has low viability after thawing. The first pregnancy obtained with frozen equine semen was reported five decades ago. According to Cazalez, 2020, it is very difficult to recommend a standard insemination dose for frozen semen in horses. Most scientific studies cannot control for the effect of other "confounding" factors such as processing method, mare factor, stallion factor, insemination technique, etc., making it difficult to critically compare them. Rigby et al. (2001) obtained similar pregnancy rates when comparing deep artificial insemination in the uterine horn with an endoscope and flexible pipette.(AU)

Improvement of in vitro fertilization by a tannin rich vegetal extract addition to frozen thawed boar sperm

Boar spermatozoa are very susceptible to cryopreservation injuries and, for this reason, pig remains one of the few species in which fresh semen is still preferred to thawed one for routine artificial insemination (AI). The present work evaluated the effect of supplementing boar sperm thawing medium with Silvafeed SP (SSP), a mixture of Chestnut and Quebracho wood extracts (60/40 w/w) rich in polyphenols (92.4% tannin content) on in vitro fertilization (IVF) and on the following sperm parameters: sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function and lipid peroxidation (assessed by flow cytometry) and capacitation status (immunolocalization of tyrosine phosphorylated proteins). Thawed spermatozoa were incubated 1 h at 37°C in BTS without (CTR) or with (5, 10, 20 µg/mL) SSP. After incubation sperm suspension was divided in three aliquots: one was used for IVF trials, one for sperm analysis, and the last one was capacitated for 1 h at 39°C 5% CO2 in IVF medium. Sperm motility parameters, viability, acrosome integrity, mitochondrial functionality, lipid peroxidation and tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, were not influenced by SSP. However, oocytes inseminated with thawed spermatozoa pretreated with all the different SSP concentrations presented a significant (P < 0.01) increase in penetration rate compared to CTR. In addition, 5 µg/mL SSP exerted a positive effect (P<0.05) on the total efficiency of fertilization. These results encourage the use of SSP in the thawing medium since post-thawing fertility is a limit for the large-scale use of boar frozen semen.

Improvement of in vitro fertilization by a tannin rich vegetal extract addition to frozen thawed boar sperm

Boar spermatozoa are very susceptible to cryopreservation injuries and, for this reason, pig remains one of the few species in which fresh semen is still preferred to thawed one for routine artificial insemination (AI). The present work evaluated the effect of supplementing boar sperm thawing medium with Silvafeed SP (SSP), a mixture of Chestnut and Quebracho wood extracts (60/40 w/w) rich in polyphenols (92.4% tannin content) on in vitro fertilization (IVF) and on the following sperm parameters: sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function and lipid peroxidation (assessed by flow cytometry) and capacitation status (immunolocalization of tyrosine phosphorylated proteins). Thawed spermatozoa were incubated 1 h at 37°C in BTS without (CTR) or with (5, 10, 20 µg/mL) SSP. After incubation sperm suspension was divided in three aliquots: one was used for IVF trials, one for sperm analysis, and the last one was capacitated for 1 h at 39°C 5% CO2 in IVF medium. Sperm motility parameters, viability, acrosome integrity, mitochondrial functionality, lipid peroxidation and tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, were not influenced by SSP. However, oocytes inseminated with thawed spermatozoa pretreated with all the different SSP concentrations presented a significant (P < 0.01) increase in penetration rate compared to CTR. In addition, 5 µg/mL SSP exerted a positive effect (P<0.05) on the total efficiency of fertilization. These results encourage the use of SSP in the thawing medium since post-thawing fertility is a limit for the large-scale use of boar frozen semen.(AU)

Influence of the Addition of Vitamin E in Extenders in the Quality of Fresh Semen Diluted, Refrigerated and Frozen Semen in Dogs of French Bulldog Breed

Background: Researches have been conducted in order to maintain the quality of the fresh semen which is diluted, refrigerated or frozen in liquid nitrogen for artificial insemination purposes in dogs. The semen biotechnology cooperates with the development of new formulations of types of extenders which minimize the death of the sperms due to thermal stress during temperature reduction of the refrigeration and freezing curves of the semen. The objective was to study the influence of the addition of vitamin E in types of extenders in the quality of the fresh, refrigerated and frozen semen in dogs of French Bulldog breed.Materials, Methods & Results: Semen samples by digital manipulation were performed on 5 adult dogs, French bulldog breed, five on each dog, totaling 25 ejaculated. The characteristics evaluated in fresh semen were: Volume (mL), color, aspect, concentration (x106/mL), sperm motility (%), vigor (1-5) and sperm morphology (%). For refrigerated and frozen semen, motility (%), vigor (1-5) and morphology (%) were analyzed. The ejaculated ones were fractionated in 4 equal parts and diluted in the ratio 1: 1 in the following extenders: 1 TRIS - Fructose Citric acid + 200 mM of vitamin E; 2 TRIS - Fructose Citric acid; 3 - coconut water (ACP-106®) + 200 mM of vitamin E; and 4 coconut water (ACP-106®). The four aliquots of semen, diluted in the four respective ex

Effect of Oral Administration of Selenium and Vitamin E on the Quality of Fresh, Refrigerated and Frozen Semen in French Bulldog Breed Dogs

Background: New methodologies have been developed seeking to maximize pregnancy rate in female dogs created in commercial kennels, and also in order to maintain the quality of canine semen after dilution, refrigeration or freezing. One of the main factors that generate damage to sperm is oxidative stress, to minimize sperm damage, selenium and antioxidants like vitamin E are administered, by oral administration, seeking to improve the quality of semen. The objective was to study the effect of vitamin E and selenium, by oral administration, in the quality of fresh, refrigerated and frozen semen in adult dogs French Bulldog breed.Materials, Methods & Results: Semen samples were collected from 5 adult dogs, French Bulldog breed, being 2 semen drawing before the daily oral supplementation with vitamin E and selenium (ESE®) and semen drawing at 20, 40 and 60 days after the beginning of oral supplement. The ejaculated samples were diluted in TRIS - fructose citric acid (3.28 g TRIS-hydroxy-methyl-amino-methane, 1.78 g of citric acid monohydrate and 1.25 g of D - fructose, dissolved in 100 mL of distilled water and added of 20% egg yolk and 6% of glycerol. The characteristics evaluated in fresh semen were: volume (mL), color, appearance, concentration (x106 / mL), sperm motility (%), sperm strength (1 to 5) and morphology (%). For refrigerated and frozen semen were analyzed: sperm

Effect of honey supplementation on Egyptian buffalo semen

This study testified the effect of honey supplementation (0.5-4.0%) in milk on the quality of chilled and frozen buffalo spermatozoa. Semen was chilled with/without honey and examined for motility, viability, plasma membranes integrity by hypo-osmotic swelling test (HOS) at 0, 1, 2 and 4 h. Frozen-thawed semen was examined for the same criteria beside the viability index and in vitro cleavage rate. The motility, livability and HOS of chilled semen upsurge with honey supplementation 1.0-2.0%. The normality of chilled spermatozoa was improved in the presence of 2.0-4.0% of honey at 4 h. Tail abnormalities decreased with milk honey 0.5, 1.0 and 2.0% at 2, 1 and 4 h, respectively. Incorporation of honey in milk extenders at levels of 0.5-2.0% was associated with an enhanced post equilibration motility. The post-thawing motility showed a steady increase with honey levels. The viability index increased (P < 0.001) with milk-honey 2.0% (109.00 ± 9.91) and 4.0% (112.00 ± 14.41). In vitro cleavage rate was clearly (P 0.001) enhanced in the co-existence of milk-honey 2.0% compared with control (74.00 vs. 45.83). In the meantime, a reasonable high cleavage rate (67.00%) was encountered with milk honey 0.5%. In conclusion, incorporation of honey in skim milk extenders is promising to enhance the characteristics and fertilizing potential of stored buffalo’s semen due to its nutritive and protective properties.

Influence of the addition of vitamin E in extenders in the quality of fresh semen diluted, refrigerated and frozen semen in dogs of French Bulldog breed / Influência da adição de vitamina E em meios diluentes na qualidade do sêmen fresco diluído, refrigerado e congelado em cães da raça Bulldog Francês

Background: Researches have been conducted in order to maintain the quality of the fresh semen which is diluted, refrigerated or frozen in liquid nitrogen for artificial insemination purposes in dogs. The semen biotechnology cooperates with the development of new formulations of types of extenders which minimize the death of the sperms due to thermal stress during temperature reduction of the refrigeration and freezing curves of the semen. The objective was to study the influence of the addition of vitamin E in types of extenders in the quality of the fresh, refrigerated and frozen semen in dogs of French Bulldog breed. Materials, Methods & Results: Semen samples by digital manipulation were performed on 5 adult dogs, French bulldog breed, five on each dog, totaling 25 ejaculated. The characteristics evaluated in fresh semen were: Volume (mL), color, aspect, concentration (x106/mL), sperm motility (%), vigor (1-5) and sperm morphology (%). For refrigerated and frozen semen, motility (%), vigor (1-5) and morphology (%) were analyzed. The ejaculated ones were fractionated in 4 equal parts and diluted in the ratio 1: 1 in the following extenders: 1 TRIS - Fructose Citric acid + 200 mM of vitamin E; 2 TRIS - Fructose Citric acid; 3 - coconut water (ACP-106®) + 200 mM of vitamin E; and 4 coconut water (ACP-106®). The four aliquots of semen, diluted in the four respective extenders were centrifuged at 1500 g/10 min and the “pellets” formed of sperm from every ejaculated, detached from the tubes wall were diluted homogeneously with the four extenders to the volume of 1.5 mL and filled into 0.5 mL French straws kept under refrigeration at 5oC/4 h after placed in a nitrogen vapor at -120ºC/15 min, and immersed in liquidnitrogen at -196ºC in the sequence stored in identified racks and stored in liquid nitrogen container until the time of thawing in a water bath at 37ºC/30 s for microscopic semen analysis. [...]

Effect of honey supplementation on Egyptian buffalo semen

This study testified the effect of honey supplementation (0.5-4.0%) in milk on the quality of chilled and frozen buffalo spermatozoa. Semen was chilled with/without honey and examined for motility, viability, plasma membranes integrity by hypo-osmotic swelling test (HOS) at 0, 1, 2 and 4 h. Frozen-thawed semen was examined for the same criteria beside the viability index and in vitro cleavage rate. The motility, livability and HOS of chilled semen upsurge with honey supplementation 1.0-2.0%. The normality of chilled spermatozoa was improved in the presence of 2.0-4.0% of honey at 4 h. Tail abnormalities decreased with milk honey 0.5, 1.0 and 2.0% at 2, 1 and 4 h, respectively. Incorporation of honey in milk extenders at levels of 0.5-2.0% was associated with an enhanced post equilibration motility. The post-thawing motility showed a steady increase with honey levels. The viability index increased (P < 0.001) with milk-honey 2.0% (109.00 ± 9.91) and 4.0% (112.00 ± 14.41). In vitro cleavage rate was clearly (P 0.001) enhanced in the co-existence of milk-honey 2.0% compared with control (74.00 vs. 45.83). In the meantime, a reasonable high cleavage rate (67.00%) was encountered with milk honey 0.5%. In conclusion, incorporation of honey in skim milk extenders is promising to enhance the characteristics and fertilizing potential of stored buffalos semen due to its nutritive and protective properties.(AU)

Influence of the addition of vitamin E in extenders in the quality of fresh semen diluted, refrigerated and frozen semen in dogs of French Bulldog breed / Influência da adição de vitamina E em meios diluentes na qualidade do sêmen fresco diluído, refrigerado e congelado em cães da raça Bulldog Francês

Background: Researches have been conducted in order to maintain the quality of the fresh semen which is diluted, refrigerated or frozen in liquid nitrogen for artificial insemination purposes in dogs. The semen biotechnology cooperates with the development of new formulations of types of extenders which minimize the death of the sperms due to thermal stress during temperature reduction of the refrigeration and freezing curves of the semen. The objective was to study the influence of the addition of vitamin E in types of extenders in the quality of the fresh, refrigerated and frozen semen in dogs of French Bulldog breed. Materials, Methods & Results: Semen samples by digital manipulation were performed on 5 adult dogs, French bulldog breed, five on each dog, totaling 25 ejaculated. The characteristics evaluated in fresh semen were: Volume (mL), color, aspect, concentration (x106/mL), sperm motility (%), vigor (1-5) and sperm morphology (%). For refrigerated and frozen semen, motility (%), vigor (1-5) and morphology (%) were analyzed. The ejaculated ones were fractionated in 4 equal parts and diluted in the ratio 1: 1 in the following extenders: 1 TRIS - Fructose Citric acid + 200 mM of vitamin E; 2 TRIS - Fructose Citric acid; 3 - coconut water (ACP-106®) + 200 mM of vitamin E; and 4 coconut water (ACP-106®). The four aliquots of semen, diluted in the four respective extenders were centrifuged at 1500 g/10 min and the “pellets” formed of sperm from every ejaculated, detached from the tubes wall were diluted homogeneously with the four extenders to the volume of 1.5 mL and filled into 0.5 mL French straws kept under refrigeration at 5oC/4 h after placed in a nitrogen vapor at -120ºC/15 min, and immersed in liquidnitrogen at -196ºC in the sequence stored in identified racks and stored in liquid nitrogen container until the time of thawing in a water bath at 37ºC/30 s for microscopic semen analysis. [...](AU)

Effect of oral administration of selenium and vitamin E on the quality of fresh, refrigerated and frozen semen in French Bulldog breed dogs / Efeito da administração via oral de selênio e vitamina E na qualidade do sêmen fresco, refrigerado e congelado em cães da raça Bulldog Francês

Background: New methodologies have been developed seeking to maximize pregnancy rate in female dogs created in commercial kennels, and also in order to maintain the quality of canine semen after dilution, refrigeration or freezing. One of the main factors that generate damage to sperm is oxidative stress, to minimize sperm damage, selenium and antioxidants like vitamin E are administered, by oral administration, seeking to improve the quality of semen. The objective was to study the effect of vitamin E and selenium, by oral administration, in the quality of fresh, refrigerated and frozen semen in adult dogs French Bulldog breed. Materials, Methods & Results: Semen samples were collected from 5 adult dogs, French Bulldog breed, being 2 semen drawing before the daily oral supplementation with vitamin E and selenium (ESE®) and semen drawing at 20, 40 and 60 days after the beginning of oral supplement. The ejaculated samples were diluted in TRIS - fructose citric acid (3.28 g TRIS-hydroxy-methyl-amino-methane, 1.78 g of citric acid monohydrate and 1.25 g of D - fructose, dissolved in 100 mL of distilled water and added of 20% egg yolk and 6% of glycerol. The characteristics evaluated in fresh semen were: volume (mL), color, appearance, concentration (x106 / mL), sperm motility (%), sperm strength (1 to 5) and morphology (%). For refrigerated and frozen semen were analyzed: [...](AU)

Effect of oral administration of selenium and vitamin E on the quality of fresh, refrigerated and frozen semen in French Bulldog breed dogs / Efeito da administração via oral de selênio e vitamina E na qualidade do sêmen fresco, refrigerado e congelado em cães da raça Bulldog Francês

Background: New methodologies have been developed seeking to maximize pregnancy rate in female dogs created in commercial kennels, and also in order to maintain the quality of canine semen after dilution, refrigeration or freezing. One of the main factors that generate damage to sperm is oxidative stress, to minimize sperm damage, selenium and antioxidants like vitamin E are administered, by oral administration, seeking to improve the quality of semen. The objective was to study the effect of vitamin E and selenium, by oral administration, in the quality of fresh, refrigerated and frozen semen in adult dogs French Bulldog breed. Materials, Methods & Results: Semen samples were collected from 5 adult dogs, French Bulldog breed, being 2 semen drawing before the daily oral supplementation with vitamin E and selenium (ESE®) and semen drawing at 20, 40 and 60 days after the beginning of oral supplement. The ejaculated samples were diluted in TRIS - fructose citric acid (3.28 g TRIS-hydroxy-methyl-amino-methane, 1.78 g of citric acid monohydrate and 1.25 g of D - fructose, dissolved in 100 mL of distilled water and added of 20% egg yolk and 6% of glycerol. The characteristics evaluated in fresh semen were: volume (mL), color, appearance, concentration (x106 / mL), sperm motility (%), sperm strength (1 to 5) and morphology (%). For refrigerated and frozen semen were analyzed: [...]

Adição de coenzima Q10 e melatonina em diluente para congelação de sêmen ovino

Durante a congelação seminal o estresse oxidativo pode influenciar negativamente a qualidade espermática. O objetivo desse experimento foi observar o efeito da adição de Coenzima Q10 (CoQ10) e Melatonina no diluente seminal sobre a qualidade espermática e taxa de prenhez em ovinos. Experimento 1: Foram utilizados o ejaculado de 2 carneiros mestiços, em 5 repetições, formando 7 tratamentos: Controle (diluente puro), C1 (0,175mM de CoQ10), C3 (0,35mM de CoQ10), C7 (0,7mM de CoQ10), M0,5 (0,5mM de melatonina), M1 (1mM de melatonina), M2 (2mM de melatonina). O sêmen foi avaliado pelo sistema computadorizado de análise espermática (CASA) nos momentos 0, 1, 2 e 3h, considerando motilidade total (MT), motilidade progressiva (MP) e porcentagem de espermatozoides rápidos (RAP). Experimento 2: Foram utilizados os ejaculados de 8 carneiros da raça Dorper, em triplicata, testando-se 4 tratamentos: Controle (diluente puro), C1 (0,175mM de CoQ10), C3 (0,35mM de CoQ10) e C7 (0,7mM de CoQ10). Foi realizada análise espermática completa pós-descongelação pelo CASA e análise de integridade e estabilidade de membranas plasmática e acrossomal, peroxidação lipídica, potencial mitocondrial e produção de ânions superóxido por citometria de fluxo, nos momentos 0 e 2h. Foram inseminadas 198 ovelhas por meio de laparoscopia, divididas em dois tratamentos: controle (n=98) e C7 (n=100), com posterior diagnóstico de gestação. No experimento 1, todos os tratamentos foram estatisticamente inferiores ao controle para o parâmetro MT. Para os parâmetros MP e RAP, C1 mostrou-se estatisticamente igual ao controle, e os demais tratamentos foram estatisticamente inferiores, quando comparados ao controle. Todos os tratamentos de Melatonina foram significativamente inferiores, para todos os parâmetros avaliados, em todos os momentos. Com relação aos momentos, foi observado diferença entre todos os momentos de avaliação, para todos os parâmetros com queda linear entre os momentos. Conclui-se que os tratamentos de CoQ10 foram ligeiramente superiores aos de Melatonina. No experimento 2, os parâmetros MT, VAP e RAP do controle demonstraram diferença estatística (p<0,05) entre os momentos 0h e 2h. C3 mostrou-se superior estatisticamente para o parâmetro VAP, analisado pelo CASA. Os animais 4, 7 e 8 demonstraram-se superiores para os parâmetros MT, MP e RAP, sendo selecionados para a etapa de inseminação artificial. No momento 0h da citometria de fluxo, C7 demonstrou maior porcentagem de células com alto potencial mitocondrial (p<0,05). No momento 2h, C1 e C7 foram estatisticamente superiores e C3 inferior, comparados ao controle. Foi observada maior taxa de prenhez para C7 (52%) quando comparado ao controle (38%). Conclui-se que a CoQ10 é capaz de proteger a célula espermática e, consequentemente, aumentar a taxa de prenhez em ovinos.

Oxidative stress can affect sperm quality during freezing. The aim of this experiment was to observe the effect of Coenzyme Q10 (CoQ10) and Melatonin addition in seminal extender on the sperm quality and pregnancy rate in sheep. Experiment 1: Ejaculates of 2 crossbred rams were used in 5 repetitions, forming 7 treatments: Control (pure seminal extender), C1 (0,175mM of CoQ10), C3 (0,35mM of CoQ10), C7 (0,7mM of CoQ10), M0,5 (0,5mM of melatonin), M1 (1mM of melatonin), M2 (2mM of melatonin). Semen was evaluated by the computer-assisted sperm analysis (CASA) at 0, 1, 2 and 3h, considering total motility (TM), progressive motility (PM) and percentage of rapid spermatozoa (PRS). Experiment 2: Ejaculates of 8 Dorper rams were used in triplicate, testing 4 treatments: Control (pure seminal extender), C1 (0,175mM of CoQ10), C3 (0,35mM of CoQ10) and C7 (0,7mM of CoQ10). Complete sperm analysis was performed after thawing by CASA and analysis of integrity and stability of plasma and acrosomal membranes, lipid peroxidation, mitochondrial potential and super oxide anions production by flow cytometry at 0 and 2h. Altogether, 198 ewes were inseminated by laparoscopy, divided into two treatments: control (n=98) e C7 (n=100), with subsequent pregnancy diagnosis. In experiment 1, all treatments were statistically inferior to control for TM parameter. Group C1 was statistically equal to control and other treatments were statistically inferior when compared to control for parameters PM and PRS. All treatments with melatonin were significantly lower, for all parameters evaluated at all times. Regarding the moments, a difference was observed between all the evaluation moments for all parameters with a linear fall between the moments. Can be concluded that CoQ10 were slightly superior to those of melatonin. In experiment 2 the parameters of TM, ATS and PRS of control group showed a statistical difference (p<0,05) between moments 0 and 2h. C3 group was statistically superior for the ATS parameter, analyzed by CASA. Animals 4, 7 and 8 were superior for the parameters TM, PM and PRS, being selected for artificial insemination stage. At moment 0h in flow cytometry, C7 group showed a higher percentage of cells with high mitochondrial potential (p<0,05). At 2h, C1 and C7 groups were statistically higher and C3 lower compared to the control. A higher pregnancy rate was observed for C7 (52%) when compared to the control (38%). It is concluded that CoQ10 is able to protect sperm cell and increase the pregnancy rate in sheep consequently.

Comparison between the conventional and automated systems for frozen cooled equine semen

This study aims to compare the efficiency of the automated system (controlled-rate freezer) and the conventional system (manual system) for freezing the equine semen after cooling at 16oC. The parameters evaluated were: motility, strength, plasmatic and acrosomal membrane integrity of spermatozoa from twelve stallions. Ejaculates from stallions were collected three times per week, during four weeks. Gel-free semen was diluted in skim milk extender and cooled at 16ºC for 24 h. After cooling, extended semen was centrifuged at 600 x g for 10 min. The supernatant was removed and sperm pellets were re-suspended using the freezing extender. Samples were then packed into 0.5 ml straws, which were divided into two parts: one for cryopreservation in a controlled-rate freezer (CR) and the other for a manual system (MS). In this study, CR showed higher values for motility (44.6%), viability (57.9%) and plasmatic membrane integrity (29.3%) when compared with MS (20, 35.7 and 5.1%), (P < 0.05), respectively, after 24 h of cooling at 16ºC. The automated system for cryopreservation of cooled semen at 16°C for 24 h was more efficient, with higher values of motility, viability and plasmatic membrane integrity when compared with the manual system.

Comparison between the conventional and automated systems for frozen cooled equine semen

This study aims to compare the efficiency of the automated system (controlled-rate freezer) and the conventional system (manual system) for freezing the equine semen after cooling at 16oC. The parameters evaluated were: motility, strength, plasmatic and acrosomal membrane integrity of spermatozoa from twelve stallions. Ejaculates from stallions were collected three times per week, during four weeks. Gel-free semen was diluted in skim milk extender and cooled at 16ºC for 24 h. After cooling, extended semen was centrifuged at 600 x g for 10 min. The supernatant was removed and sperm pellets were re-suspended using the freezing extender. Samples were then packed into 0.5 ml straws, which were divided into two parts: one for cryopreservation in a controlled-rate freezer (CR) and the other for a manual system (MS). In this study, CR showed higher values for motility (44.6%), viability (57.9%) and plasmatic membrane integrity (29.3%) when compared with MS (20, 35.7 and 5.1%), (P < 0.05), respectively, after 24 h of cooling at 16ºC. The automated system for cryopreservation of cooled semen at 16°C for 24 h was more efficient, with higher values of motility, viability and plasmatic membrane integrity when compared with the manual system.(AU)

Detecção de Tritrichomonas foetus em Sêmen Bovino Criopreservado através de Isolamento e Reação em Cadeia da Polimerase

A Tricomonose Genital Bovina (TGB), uma doença infectocontagiosa causada pelo protozoário flagelado Tritrichomonas foetus, transmitida por via venérea e por fômites contaminados, possui grande importância econômica, pois sua principal consequência é o aborto no terço inicial da gestação, além de produzir infertilidade temporária. Em machos a doença é assintomática e permanente, caracterizando os touros como portadores silenciosos e ressaltando o direcionamento do diagnóstico nesta categoria de animais. A identificação e detecção do protozoário por meio de cultivo são tidas como padrão ouro dentre as técnicas diagnósticas nas centrais de inseminação artificial brasileiras. Entretanto, há a possibilidade de resultado falso negativo, sendo necessário o desenvolvimento de técnicas mais sensíveis e eficazes de diagnóstico já que, até o momento, a Inseminação Artificial (IA) é a principal forma de prevenção da TGB. T. foetus já foi encontrado em amostras de sêmen e também é capaz de permanecer viável quando congelado, o que eleva a IA a um potencial fator de risco quando medidas sanitárias não são aplicadas ou o método diagnóstico é insuficiente. Com base no exposto, este trabalho teve por objetivo investigar a presença de T. foetus em 27 amostras de sêmen bovino congelado, por meio de cultivo e Reação em Cadeia da Polimerase (PCR). Uma vez no laboratório, as amostras foram descongeladas em água a 37ºC, parte foi inoculada em tubo de ensaio contendo meio Diamond, incubada a 35ºC com consequente avaliação de crescimento e avaliadas a cada 24 horas, via exame direto em microscópio, e a outra parte foi diluída em PBS para posterior análise molecular. Após 10 dias de cultivo, todas as amostras foram negativas. Para a detecção molecular foi utilizado o Kit Quick-DNA Miniprep (Zymo Research) sem proteinase K para extração do DNA. Os iniciadores utilizados na PCR foram TRF3 e TRF4. O resultado da PCR foi negativo para todas as amostras, respaldando o resultado obtido no cultivo. Conclui-se que as amostras utilizadas foram realmente negativas para a presença do patógeno pesquisado em ambos os métodos diagnósticos, o que comprovou a inocuidade do sêmen comercial testado.

The Bovine Genital Trichomoniasis (BGT) is an infectious disease triggered by a protozoan called Tritrichomonas foetus, which is transmitted by venereal route and by contaminated fomites. The disease is economically relevant, since its main consequence is abortion, besides producing temporary infertility. In males the disease is asymptomatic and permanent, characterizing the bulls as silent nurses and emphasizing the direction of diagnosis in this category of animals. The identification and detection of the protozoan by microbiological culture are considered as "gold standard" among diagnostic techniques in Brazilian artificial insemination centers. However, the possibility of a false negative is a reality and is needed more sensitive and specific techniques of diagnose, since the Artificial Insemination (AI) is the current way to prevent BGT. T. foetus has already been found in semen samples and is also able to remain viable when frozen, raising AI to a risk factor when sanitary measures are not applied or the diagnostic method is insufficient Thus, this study is aimed to detect the presence of T. foetus in samples of cryopreserved bovine semen, through culture and Polymerase Chain Reaction (PCR). The semen straws will be purchased from artificial insemination centers. A total of 27 samples were analyzed. Once in the laboratory, they were thawed in water at 37ºC, part was inoculated in a test tube containing Diamond medium, incubated at 35ºC with consequent growth evaluation and evaluated every 24 hours via direct microscope examination and the other part diluted in PBS for subsequent molecular analysis. After 10 days of cultivation, all samples were negative. For molecular detection, the Quick-DNA Miniprep Kit (Zymo Research) without proteinase K was used for DNA extraction. The primers used in PCR were TRF3 and TRF4. The PCR result was negative for all samples, corroborating with that obtained in the culture. It was concluded that the samples used were negative for the presence of T. foetus in both diagnostic methods, proving the innocuousness of the commercial semen tested.

UTILIZAÇÃO DE DIFERENTES DILUENTES NA CRIOPRESERVAÇÃO DE ESPERMATOZOIDES DE GARANHÕES MANGALARGA MARCHADOR

title>Abstract /title> p>The techniques applied to animal reproduction such as artificial insemination, transfer and italic>in vitro /italic> production of embryos, heat synchronization and induction, and gametes cryopreservation have been more utilized in veterinary practice each day. Nevertheless, some techniques have not achieved their full technical capacity within the equine reproduction field, such as semen cryopreservation. The aim of this study was to evaluate the characteristics of post-thawing semen (total motility, strength, plasmatic and acrosomal membrane integrity) of Mangalarga Marchador breed stallions, using three different extenders (Botucrio, FR5 and FR6). Ejaculates from five stallions were collected and the gel-free semen was diluted in a 1:1 dilution in skim milk extender, and centrifuged at 600 g for 10 minutes. After the centrifugation the supernatant was removed and sperm pellet was divided and re-suspended using three different extenders to a concentration of 200 x 10 sup>6 /sup> cells/mL. The samples were packed into 0.5 mL straws, placed in a stainless steel support and kept inside the refrigerator (5 ºC) for one hour. Subsequently, these straws were kept at a height of 6 cm from liquid nitrogen for 15 minutes in an isotherm box and, after that, plunged into liquid nitrogen (-196 ºC) and stored in a liquid nitrogen holding tank. There were no differences in the parameters evaluated when extenders using mixed amides and glycerol (Botucrio and FR6) were used (P > 0.05). All parameters evaluated were lower for the extender containing only glycerol (P 0.05). The use of cryoprotectants (methylformamide and dimethylformamide) in association with glycerol concentrations around 1 to 2% is an alternative for semen cryopreservation of Mangalarga Marchador breed stallions. /p>

title>Resumo /title> p>As biotécnicas aplicadas à reprodução animal como inseminação artificial, transferência e produção italic>in vitro /italic> de embriões, indução e sincronização de cio e congelamento de gametas vêm sendo cada vez mais utilizadas na prática veterinária. No entanto, algumas biotécnicas ainda não alcançaram o seu total aperfeiçoamento técnico na reprodução equina, como a criopreservação de sêmen. Objetivou-se avaliar as características pós-descongelamento (motilidade total, vigor e integridade de membrana plasmática e acrosomal) dos espermatozoides de garanhões da raça Mangalarga Marchador (n=5), empregando-se três diluentes de criopreservação. Após a colheita, o sêmen foi diluído na proporção de 1:1 em meio à base de leite em pó desnatado e centrifugado a 600 G por 10 minutos. Após a centrifugação, o sobrenadante foi desprezado e o italic>pellet /italic> obtido dividido e ressuspendido com Botucrio, FR5 ou FR6. As amostras foram envasadas em palhetas de 0,5 mL sendo a concentração ajustada para 200x10 sup>6 /sup>espermatozoides/mL. As palhetas foram distribuídas em uma plataforma-suporte e estabilizadas a 5 ºC/60 min., em refrigerador comercial. Para o congelamento, as palhetas, posicionadas horizontalmente, foram expostas por 15 minutos ao vapor de nitrogênio líquido, em uma caixa de isopor, a 6 cm acima do nível de nitrogênio líquido. Logo em seguida, as palhetas foram imersas no nitrogênio líquido, acondicionadas em raques e estocadas em botijão criogênico a -196 ºC, para posterior avaliação. Não houve diferença para as variáveis motilidade, vigor e integridade das membranas plasmática e acrossomais quando se utilizaram diluentes que contêm a associação de amidas e glicerol (Botucrio e FR6; P>0,05). As variáveis seminais no diluente contendo apenas glicerol foram inferiores em todas as avaliações (P 0,05). A utilização de crioprotetores como a metilformamida, em associação com concentrações de 1 ou 2% de glicerol é uma alternativa para a criopreservação do sêmen de garanhões da raça Mangalarga Marchador. /p>

Biotecnologias de seleção, conservação e multiplicação de recursos genéticos de ovinos Santa Inês

The preservation of animal genetic biodiversity is fundamental to food security. Santa Ines (SI) is an important native Brazilian hair sheep breed that should be preserved by biotechnologies of selection, conservation and multiplication. The molecular biotechnology identify genes of interest that have an impact on the reproductive, productive and health performance, adding value to these animals, while reproduction biotechnologies, especially semen cryopreservation and artificial insemination, are used for multiplication, conservation and dissemination of this germplasm. Activities related to the conservation and adding value to SI should continue to be stimulated otherwise an irretrievable loss of its originality and genetic diversity can happen.

A preservação da biodiversidade genética dos animais é fundamental para a segurança alimentar. O ovino Santa Inês (SI) é um importante patrimônio genético autóctone brasileiro que deve ser conservado através do uso de biotecnologias para seleção, conservação e multiplicação. As biotecnologias moleculares identificam genes de interesse que têm impacto sobre o desempenho reprodutivo, produtivo e sanitário, agregando valores a estes animais, enquanto que, as biotecnologias da reprodução, em especial a criopreservação do sêmen e inseminação artificial, são utilizadas para a multiplicação, conservação e disseminação deste germoplasma. As atividades relacionadas à conservação e agregação de valores ao ovino SI devem continuar sendo estimuladas sob pena de uma irrecuperável perda da sua originalidade e diversidade genética.

Utilização de diferentes diluentes na criopreservação de espermatozoides de garanhões mangalarga marchador / Use of different extenders to cryopreservation of Mangalarga Marchador sperm

The techniques applied to animal reproduction such as artificial insemination, transfer and in vitro production of embryos, heat synchronization and induction, and gametes cryopreservation have been more utilized in veterinary practice each day. Nevertheless, some techniques have not achieved their full technical capacity within the equine reproduction field, such as semen cryopreservation.  The aim of this study was to evaluate the characteristics of post-thawing semen (total motility, strength, plasmatic and acrosomal membrane integrity) of Mangalarga Marchador breed stallions, using three different extenders (Botucrio, FR5 and FR6).  Ejaculates from five stallions were collected and the gel-free semen was diluted in a 1:1 dilution in skim milk extender, and centrifuged at 600 g for 10 minutes. After the centrifugation the supernatant was removed and sperm pellet was divided and re-suspended using three different extenders to a concentration of 200 x 106 cells/mL. The samples were packed into 0.5 mL straws, placed in a stainless steel support and kept inside the refrigerator (5 oC) for one hour. Subsequently, these straws were kept at a height of 6 cm from liquid nitrogen for 15 minutes in an isotherm box and, after that, plunged into liquid nitrogen (-196 oC) and stored in a liquid nitrogen holding tank. There were no differences in theparameters evaluated when extenders using mixed amides and glycerol (Botucrio and FR6) were used (P > 0.05). All parameters evaluated were lower for the extender containing only glycerol (P<0.05). The use of cryoprotectants (methylformamide and (imethylformamide) in associationwith glycerol concentrations around 1 to 2% is an alternative for semen cryopreservation of Mangalarga Marchador breed stallions.

As biotécnicas aplicadas à reprodução animal como inseminação artificial, transferência e produção in vitro de embriões, indução e sincronização de cio e congelamento de gametas vêm sendo cada vez mais utilizadas na prática veterinária. No entanto, algumas biotécnicas ainda não alcançaram o seu total aperfeiçoamento técnico na reprodução equina, como a criopreservação de sêmen. Objetivou-se avaliar as características pós-descongelamento (motilidade total, vigor e integridade de membrana plasmática e acrosomal) dos espermatozoides de garanhões da raça Mangalarga Marchador (n=5), empregando-se três diluentes de criopreservação. Após a colheita, o sêmen foi diluído na proporção de 1:1 em meio à base de leite em pó desnatado e centrifugado a 600 G por 10 minutos. Após a centrifugação, o sobrenadante foi desprezado e o pellet obtido dividido e ressuspendido com Botucrio, FR5 ou FR6. As amostras foram envasadas em palhetas de 0,5 mL sendo a concentração ajustada para 200x106 espermatozoides/mL. As palhetas foram distribuídas em uma plataforma-suporte e estabilizadas a 5 ºC/60 min., em refrigerador comercial. Para o congelamento, as palhetas, posicionadas horizontalmente, foram expostas por 15 minutos ao vapor de nitrogênio líquido, em uma caixa de isopor, a 6 cm acima do nível de nitrogênio líquido. [...]

Cryopreserved sperm for oocyte fertilization of dourado Salminus brasiliensis / Sêmen criopreservado na fertilização de ovócitos de dourado Salminus brasiliensis

Salminus brasiliensis is a migratory fish that has attracted considerable interest for aquaculture. Several procedures for induced spawning are known; however, there is a lack of protocol which enables the use of cryopreserved semen. Therefore, this study was conducted to investigate the use of cryopreserved semen using different volumes of cryopreserved semen relative to oocytes, different activators solutions and different maintenance time during the fertilization of dourado to evaluate the impact of these parameters on the fertilization rate. The semen was collected, cryopreserved in 0.5mL straws and stored in a dry shipper. Oocytes samples were fertilized according to each treatment. The different activator solutions and the contact times of the gametes with activators affected significantly the fertilization rates, which ranged between 13.4 and 27.8%, while fresh semen fertility rate was 80.8%. The relationship between oocyte and cryopreserved semen was significant, being the best ratio 0.05mL of cryopreserved semen per 10g of oocytes, while upper or lower volumes promoted a reduction in fertilization. The use of cryopreserved semen was effective to fertilize S. brasiliensis oocytes, however produced lower fertility rate than fresh semen.(AU)

O dourado, Salminus brasiliensis, é um peixe migrador que tem despertado interesse para piscicultura. Os procedimentos convencionais para a sua reprodução são conhecidos, contudo, falta um protocolo para o uso do sêmen criopreservado. Dessa forma, objetivou-se avaliar diferentes volumes de sêmen criopreservado, ouso de diferentes soluções ativadoras e com diferentes tempos de contato do sêmen com os ovócitos na taxa de fertilização. Para tal, o sêmen foi coletado e criopreservado em palhetas de 0,5mL em vapor de nitrogênio líquido. Amostras de ovócitos foram fertilizadas conforme os distintos tratamentos. As diferentes soluções testadas e o tempo de contato dos ativadores afetaram significativamente as taxas de fertilização, com valores que variaram entre 13,4 e 27,8%, enquanto o sêmen fresco propiciou 80,8% de taxa de fertilização. O volume de sêmen criopreservado afetou a taxa de fertilização dos ovos, sendo 0,05mL para 10g de ovócitos, o que promoveu os melhores resultados, sendo que volumes superiores e inferiores promoveram redução na fertilização. O uso de sêmen criopreservado foi efetivo na fertilização dos ovócitos de dourado, no entanto, foram obtidas taxas de fertilização inferiores àquelas observadas com o uso de sêmen fresco.(AU)

Biotecnologias de seleção, conservação e multiplicação de recursos genéticos de ovinos Santa Inês

The preservation of animal genetic biodiversity is fundamental to food security. Santa Ines (SI) is an important native Brazilian hair sheep breed that should be preserved by biotechnologies of selection, conservation and multiplication. The molecular biotechnology identify genes of interest that have an impact on the reproductive, productive and health performance, adding value to these animals, while reproduction biotechnologies, especially semen cryopreservation and artificial insemination, are used for multiplication, conservation and dissemination of this germplasm. Activities related to the conservation and adding value to SI should continue to be stimulated otherwise an irretrievable loss of its originality and genetic diversity can happen.(AU)

A preservação da biodiversidade genética dos animais é fundamental para a segurança alimentar. O ovino Santa Inês (SI) é um importante patrimônio genético autóctone brasileiro que deve ser conservado através do uso de biotecnologias para seleção, conservação e multiplicação. As biotecnologias moleculares identificam genes de interesse que têm impacto sobre o desempenho reprodutivo, produtivo e sanitário, agregando valores a estes animais, enquanto que, as biotecnologias da reprodução, em especial a criopreservação do sêmen e inseminação artificial, são utilizadas para a multiplicação, conservação e disseminação deste germoplasma. As atividades relacionadas à conservação e agregação de valores ao ovino SI devem continuar sendo estimuladas sob pena de uma irrecuperável perda da sua originalidade e diversidade genética.(AU)