Sperm cell capacitation status of ram semen after cooling

Background: The use of conventional artificial insemination (AI) in sheep production is usually associated with lower fertility rates when frozen semen is used. Cooled ram semen has been an alternative over frozen semen due to the higher viability, seminal quality and fertility rates following AI. The semen preservation process promotes sperm cell modifica¬tions similar to capacitation (capacitation-like) that causes cell damage affecting viability and seminal quality, but such effects are unclear for cooled semen. The aim of this study was to determine the status of sperm cell capacitation (CA) and acrosome reaction (AR) during ram semen processing and cooling under different extenders, dilution factors, and aerobiosis conditions as a function of storage time at 5oC. Materials, Methods & Results: Two consecutive ejaculates per day per male were collected from 2 adult rams by artifi¬cial vagina at 48-72 h intervals, in three replications. After macro- and microscopic evaluations, semen was segregated into groups under 3 extenders (Tris-egg yolk or TY, citrate-egg yolk or CY, skimmed milk or SM), 2 dilution factors (1 x 109 or Bi, 100 x 106 or Mi cells/mL), and 2 aerobiosis conditions (aerobic or A, semi-anaerobic or SA). Diluted semen was cooled to 5ºC and stored for up to 72 h, with evaluations every 24 h. Aliquots of fresh ejaculates and of each cooled diluted subgroup, according to extender, dilution, and aerobiosis, were collected at times T0 and T72 for determination of acrosome status and membrane integrity by the chlortetracycline (CTC) and trypan blue-Giemsa stainings, respectively. No differences were detected in sperm cell motility (M) and motility vigor (V) between fresh and diluted semen. After cooling, a significant decrease in M was observed after 48 h in CY and SM compared with fresh semen and 0 h of cool¬ing, while V started to decrease after 24 h in CY compared with TY. Likewise, M/V from different dilutions and aerobic conditions decreased more significantly after 48 and 24 h of cooling, respectively. The sperm capacitation status did not show differences in the proportion of non-capacitated (NCA), CA and AR sperm cells between TY, CY, and SM extend¬ers (NCA: 75.0%, 71.3%, 74.0%; CA: 15.7%, 17.2%, 15.9%; AR: 9.3%, 11.5%, 10.2%) or between Bi and Mi dilutions (NCA: 74.0%, 72.9%; CA: 15.9%, 16.6%; AR: 10.1%, 10.5%), respectively. However, differences (P < 0.05) were observed between A and SA aerobic conditions, with CA (17.0% vs. 15.5%) and AR (11.9% vs. 8.7%) rates being higher in A than SA, respectively, with no differences in NCA (71.1% vs. 75.8%), irrespective of the storage time. Sperm cell viability decreased after 48 h, especially in CY (P < 0.05). Discussion: Ram sperm cells can suffer irreversible damage due to thermal shock during cooling. Egg yolk-based extend¬ers provide phospholipids and cholesterol to protect the sperm cell membrane during the thermal shock caused by the change in temperature. In this study, sperm cells had irreversible decreases in M/V, with increase in acrosome and plasma membrane damage after cooling to 5ºC. The largest and smallest decreases in M and V over time were observed in the CY and TY extenders, respectively. In addition to the extender type, the semen preservation method and storage time promoted changes in the capacitation status, AR and in sperm cell viability, which per se were associated with a decrease in semen fertility. In fact, the proportions of CA and/or AR sperm cells gradually increased over time after dilution and storage at 5ºC, with a negative correlation between sperm cell viability and M/V over time. In summary, extender and cooling time affected mostly M/V, while aerobiosis condition and dilution factor were more associated with acrosome status and sperm survival, with the extender having less impact on the acrosome status as a function of time

Sperm cell viability and status of sperm cell capacitation of ram semen refrigerated under distinct extenders, dilution factors and aerobic conditions

O objetivo deste estudo foi de determinar o status de capacitação espermática (CA) e de reação acrossomal (RA) durante o processamento e refrigeração do sêmen ovino sob diferentes diluentes, fatores de diluição e condições de aerobiose em função do tempo. Foram coletados ejaculados de dois carneiros adultos por vagina artificial a intervalos de 48-72 h. Após avaliações macro- e microscópicas, o sêmen foi segregado em grupos sob três diluentes (Tris-gema, citrato-gema ou leite desnatado), dois fatores de diluição (1 x 109 ou 100 x 106 células/mL) e duas condições de aerobiose (aeróbico ou semi-anaeróbico). O sêmen foi mantido refrigerado por 72 h, com avaliações a cada 24 h desde o equilíbrio a 5oC (T0). Alíquotas dos ejaculados frescos e de cada sub-grupo refrigerado de acordo com o diluente, a diluição e a aerobiose, foram coletadas nos tempos T0 e T72 para a determinação do status do acrossomo por coloração com clortetraciclina (CTC), e da integridade de membrana com azul de tripano e Giemsa. As maiores e menores quedas de motilidade e vigor espermáticos (M/V) em função do tempo foram observadas com a utilização de citrato-gema e Tris-gema, respectivamente. A proporção de espermatozoides com capacitação e/ou reação acrossomal aumentou após a diluição e armazenamento do sêmen ovino a 5oC, havendo correlação negativa com a viabilidade e M/V em função do tempo. Os fatores de variação sobre a M/V foram o diluente e o tempo de refrigeração, enquanto o status do acrossomo e a sobrevivência espermática esteve mais associada à condição de aerobiose e fator de diluição, com o diluente apresentando menor impacto no status do acrossomo em função do tempo.The aim of this study was to determine the status of sperm capacitation (CA) and acrosome reaction (AR) during processing and refrigeration of ovine semen under different extenders, dilution factors and aerobiosis conditions as a function of time. Ejaculates were collected from two adult rams by artificial vagina at 48-72 h intervals. After macro- and microscopic evaluations, semen was segregated into groups under three extenders (Tris-egg yolk, citrate-egg yolk or skimmed milk), two dilution factors (1 x 109 or 100 x 106 cells/mL) and two aerobiosis conditions (aerobic or semi-anaerobic). Diluted semen was kept refrigerated for 72 h, with evaluations every 24 h from the equilibrium at 5 oC (T0). Aliquots of fresh ejaculates and of each refrigerated diluted subgroup according to extender, dilution and aerobiosis were collected at times T0 and T72 for determination of acrosome status by staining with chlortetracycline (CTC), and membrane integrity with trypan blue and Giemsa. The largest and smallest falls in sperm motility and sperm vigor (M/V) as a function of time were observed with the use of citrate-yolk and Tris-yolk extenders, respectively. The proportion of capacitated and/or acrosome reacted sperm cells gradually increased over time after dilution and storage at 5oC, with a negative correlation observed with viability and M/V as a function of time. Variation factors on M/V were extender and refrigeration time, while acrosome status and sperm survival were more associated with aerobiosis condition and dilution factor, with the extender having less impact on the acrosome status as a function of time

Antimicrobial activity of the ethanolic extract of propolis against bacteria that cause mastitis in cattle

Propolis is a substance produced by bees, especially Apis mellifera, from resins of plant shoots. It possesses numerous biological properties, such as antimicrobial and anti-inflammatory. Activities such as organic dairy farming show high demand for natural products with proprieties like those of propolis, since milk contamination and mastitis are major problems of the dairy industry. The objective of this study was to evaluate in vitro the antimicrobial activity of different concentrations of an ethanolic extract of propolis (EEP). Action of the EEP extracted from beehives in southern Brazil was tested against 13 different genera of bacterial agents that cause mastitis in dairy cattle. The experiment was carried out in the Laboratory of Parasitology and Microbiology at the Federal University of Pampa, Campus Dom Pedrito - RS, Brazil, during May and June 2016. Agar diffusion and microdilution plate methodologies were followed. Based on the results of the minimum inhibitory concentration test (MIC), the EEP had inhibitory activity on 100% of the bacteria tested at concentrations above 10% (w/v). These results show that propolis has antimicrobial potential against bacteria involved in the process of mastitis.A própolis é uma substância coletada por abelhas, especialmente pela Apis mellifera, a partir de resinas de brotos de plantas que possuem numerosas propriedades biológicas, como antimicrobiana e antiinflamatória. Atividades como a pecuária leiteira orgânica apresentam alta demanda por produtos naturais com propriedades como as presentes na própolis, uma vez que a contaminação do leite e a mastite são grandes problemas da indústria de laticínios. O objetivo deste trabalho foi avaliar a atividade antimicrobiana in vitro do extrato etanólico de própolis (EEP) em diferentes concentrações. Ação do EEP extraído de colmeias no sul do Brasil foi testada contra 13 diferentes gêneros de agentes bacterianos causadores de mastite em bovinos leiteiros. O experimento foi conduzido no laboratório de Parasitologia e Microbiologia da Universidade Federal do Pampa, Campus Dom Pedrito – RS, durante os meses de maio e junho de 2016. Seguiram-se as técnicas de difusão em ágar e microdiluição em placas. Através dos resultados do teste de concentração inibitória mínima (CIM), pode-se afirmar que o EEP possui atividade inibitória em 100% das bactérias testadas em concentrações acima de 10% (p/v). Tais resultados mostram que a própolis apresenta potencial antimicrobiano frente as principais bactérias envolvidas nos processos de mastite.