RESUMO
Sheep pain is an animal welfare issue monitored based on behavioral responses, including appetite. Dominant (alpha) males have priority for accessing limited feed resources, however, the effects of pain on feed interest in members of a group with defined social hierarchy are unknown. Our objective was to investigate effects of acute post-orchiectomy pain on alpha rams' interest in accessing a limited feed resource. Eighteen rams were randomly housed in pens of 3 rams. After acclimation, the first 5-d (consecutive) battery of a behavior test was performed. In this test, 180 g of the regular diet concentrate was placed in a portable trough in the center of the pen; this feed was supplemental to the diet and represented a limited, albeit strongly preferable feed resource. Rams were filmed for 5 min after the feed introduction. Hierarchical levels (alpha, beta, and gamma) were defined based on the social hierarchical index according to higher initiator and lower receptor agonistic behaviors from the social network analyses. After 15 d, a second 5-d behavioral test battery was repeated. On the following day, alpha rams were castrated. Flunixin meglumine was given immediately before surgery and a final behavioral test was performed 8 h post-orchiectomy, concurrent with an expected peak in postoperative pain. For all recordings, the latency, frequency, and duration of time that each ram had its mouth inside the feed trough were recorded, and the Unesp-Botucatu sheep acute pain scale pain scale (USAPS) was applied. The social hierarchical index was highest in alpha rams, followed by beta and gamma. The pain scores were statistically equivalent across the 11 evaluation days for beta and gamma rams, whereas there was an increase in the final evaluation for alpha. There was no difference in latency, frequency, and duration between alpha, beta, and gamma rams across evaluations. We concluded that acute post-orchiectomy pain did not decrease alpha rams' interest in accessing limited feed. Routine feeding offers a valuable chance to detect pain-related behavior using the USAPS in rams. However, dominance may confound appetite-related behaviors in assessing acute pain, as alpha rams' interest in limited feed remained unaffected by the pain.
RESUMO
O objetivo deste estudo foi padronizar a técnica do azul de toluidina para a análise da fragmentação do DNA de espermatozoides de touro, carneiro e garanhão. Para tanto, utilizamos seis animais de cada espécie. O esperma foi coletado e as amostras foram divididas em duas alíquotas: uma amostra de espermatozoides foi mantida a 5°C (DNA de espermatozoide intacto), e as amostras remanescentes foram submetidas à indução da fragmentação do DNA. As amostras foram então misturadas para obter proporções conhecidas e progressivas de espermatozoides com DNA fragmentado (0, 25, 50, 75 e 100%). Os esfregaços de sêmen foram realizados e submetidos à coloração com TB. Observamos altos coeficientes de regressão linear entre a proporção esperada de DNA danificado e os resultados de TB para amostras de carneiro, touro e garanhão. Em conclusão, a coloração de TB foi considerada uma técnica rápida e eficaz para o estudo do DNA dos espermatozoides.(AU)
The aim of this study was to standardize the toluidine blue stain technique for the analysis of DNA fragmentation of bull, ram and stallion spermatozoa. For this purpose, we used six animals of each specie. Sperm was collected and samples were splited into two aliquots: a sperm sample was kept at 5°C (intact sperm DNA), and the remaining samples were submitted to the induction of DNA fragmentation. Samples were then mixed to obtain known and progressive proportions of sperm with fragmented DNA (0, 25, 50, 75 and 100%). Semen smears were performed and subjected to staining with TB. We observed high linear regression coefficients between the expected proportion of damaged DNA and the results of TB for ram, bull and stallion samples. In conclusion, TB stain was considered a fast and effective technique for the study of spermatozoa DNA.
El objetivo de este estudio fue estandarizar la técnica del azul de toluidina para el análisis de la fragmentación del ADN de espermatozoides de toro, carnero y semental. Para ello, utilizamos seis animales de cada especie. El esperma fue recogido y las muestras se dividieron en dos alícuotas: una muestra de espermatozoides se mantuvo a 5 ° C (ADN de espermatozoides intacto), y las muestras restantes se sometieron a la inducción de la fragmentación del ADN. Las muestras se mezclaron para obtener proporciones conocidas y progresivas de espermatozoides con ADN fragmentado (0, 25, 50, 75 y 100%). Los frotis de semen fueron realizados y sometidos a la coloración con TB. Se observaron altos coeficientes de regresión lineal entre la proporción esperada de ADN dañado y los resultados de TB para muestras de carnero, toro y semental. En conclusión, la tinción de TB fue considerada una técnica rápida y eficaz para el estudio del ADN de los espermatozoides.(AU)
Assuntos
Animais , Masculino , Bovinos , Cloreto de Tolônio , Fragmentação do DNA , Sêmen , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/veterinária , Ovinos , Cavalos , Técnicas de Reprodução Assistida/veterináriaRESUMO
O objetivo deste estudo foi padronizar a técnica do azul de toluidina para a análise da fragmentação do DNA de espermatozoides de touro, carneiro e garanhão. Para tanto, utilizamos seis animais de cada espécie. O esperma foi coletado e as amostras foram divididas em duas alíquotas: uma amostra de espermatozoides foi mantida a 5°C (DNA de espermatozoide intacto), e as amostras remanescentes foram submetidas à indução da fragmentação do DNA. As amostras foram então misturadas para obter proporções conhecidas e progressivas de espermatozoides com DNA fragmentado (0, 25, 50, 75 e 100%). Os esfregaços de sêmen foram realizados e submetidos à coloração com TB. Observamos altos coeficientes de regressão linear entre a proporção esperada de DNA danificado e os resultados de TB para amostras de carneiro, touro e garanhão. Em conclusão, a coloração de TB foi considerada uma técnica rápida e eficaz para o estudo do DNA dos espermatozoides.
The aim of this study was to standardize the toluidine blue stain technique for the analysis of DNA fragmentation of bull, ram and stallion spermatozoa. For this purpose, we used six animals of each specie. Sperm was collected and samples were splited into two aliquots: a sperm sample was kept at 5°C (intact sperm DNA), and the remaining samples were submitted to the induction of DNA fragmentation. Samples were then mixed to obtain known and progressive proportions of sperm with fragmented DNA (0, 25, 50, 75 and 100%). Semen smears were performed and subjected to staining with TB. We observed high linear regression coefficients between the expected proportion of damaged DNA and the results of TB for ram, bull and stallion samples. In conclusion, TB stain was considered a fast and effective technique for the study of spermatozoa DNA.
El objetivo de este estudio fue estandarizar la técnica del azul de toluidina para el análisis de la fragmentación del ADN de espermatozoides de toro, carnero y semental. Para ello, utilizamos seis animales de cada especie. El esperma fue recogido y las muestras se dividieron en dos alícuotas: una muestra de espermatozoides se mantuvo a 5 ° C (ADN de espermatozoides intacto), y las muestras restantes se sometieron a la inducción de la fragmentación del ADN. Las muestras se mezclaron para obtener proporciones conocidas y progresivas de espermatozoides con ADN fragmentado (0, 25, 50, 75 y 100%). Los frotis de semen fueron realizados y sometidos a la coloración con TB. Se observaron altos coeficientes de regresión lineal entre la proporción esperada de ADN dañado y los resultados de TB para muestras de carnero, toro y semental. En conclusión, la tinción de TB fue considerada una técnica rápida y eficaz para el estudio del ADN de los espermatozoides.