Ellen Cordeiro Bento da Silva

Effect of antioxidants resveratrol and quercetin on in vitro evaluation of frozen ram sperm

The objective was to assess the effects of the antioxidants resveratrol and quercetin on frozen-thawed ram sperm. Semen samples (which exceeded minimum standards) from four mature crossbreed Santa Inês rams were pooled and aliquots of each pool were diluted in Tris-egg yolk-glycerol, with the addition of 0, 5, 10, 15, and 20 μg/mL of resveratrol and quercetin in Experiment 1 and Experiment 2, respectively. In Experiment 1, the proportion of sperm with a high mitochondrial membrane potential was greater (P < 0.02) in the control group than in resveratrol 20 μg/mL group. In Experiment 2, the proportion of sperm with high mitochondrial membrane potential was greater in the control group (P < 0.0001) than in the other experimental groups, and greater in the quercetin 5 μg/mL group (P < 0.05) than in the other quercetin-treated groups. Thus, addition of 5 to 20 μg/mL of either resveratrol or quercetin to the Tris-egg yolk-glycerol extender reduced sperm mitochondrial membrane potential.

In vitro evaluation of ram sperm frozen with glycerol, ethylene glycol or acetamide

The aim was to assess the in vitro effect of glycerol, ethylene glycol or acetamide on frozen-thawed ram spermatozoa. Aliquots of each sixteen ejaculates from four rams of the Morada Nova breed were diluted in Tris-egg yolk with glycerol (5%), ethylene glycol (3% or 5%) or acetamide (3% or 5%) and frozen at -196°C. After thawing, progressive sperm motility was greater (P<0.05) in cryopreservation with glycerol 5% and ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Acrosome integrity was greater (P<0.05) with ethylene glycol 5% than acetamide (3% or 5%). The percentage of sperm without oxidative stress was greater (P<0.05) with ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Plasma membrane integrity was greater with glycerol 5% (P<0.05) than with the other cryoprotectants. Thus, it is concluded that glycerol 5% and ethylene glycol 3% or 5% protect ram sperm against the harmful effects of freezing and that glycerol 5% offers greater protection to sperm plasma membrane.

Efeito da adição de trolox e pentoxifilina na motilidade, integridade do acrossoma e do DNA de espermatozoides equinos após descongelação

Three stallions were used to study the effect of trolox and pentoxifylline addition on the motility and integrity of acrossome and DNA equine spermatozoa after thawing. Tris-egg-yolg diluent with glycerol (5%) were used to freeze the semen samples in a freezing machine. The samples were thawed at 37oC during 30 seconds and treated with: T1=150µL of semen + 150µL of Tris; T2= 150µL of semen + 150µL of Tris +150mM/mL of trolox; T3= 150µL of semen + 150µL Tris +3.5mM of pentoxifylline; and T4= 150µL of semen + 150µL of Tris + 3.5mM of pentoxifylline + 150mM of trolox. After 0, 60, and 120 minutes of incubation (37oC), the samples were analyzed to motility, vigor, and integrity of acrossome and DNA. There was no difference (P>0.05) among treatments considering 0 and 60 minutes of incubation in all studied parameters. After 120 minutes of incubation, it was observed higher percentage (P<0.05) of cells with total and progressive motility in the samples of T2. It can be concluded that the trolox addition after thawing of equine semen preserved total and progressive motility of the sperm incubated at 37oC during 120 minutes.

Efeito da adição de glutationa peroxidase e cisteína ao diluidor de congelação do sêmen equino

Ejaculates (n=25) of horses were used to assess the effect of glutathione peroxidase (GPx) and cysteine on the viability of frozen sperm cells. Semen was extended at Botu Crio with antioxidants, and divided in groups: G1, control; G2, 1 U GPx; G3, 5U GPx; G4, 0.5mM cysteine and G5, 1mM cysteine, packed in 0.5mL straws, and frozen. After thawing (37° C for 30 seconds) samples were analyzed for plasma membrane (IMP) and acrosome integrity (IAc), mitochondrial membrane potential (MMP) and kinematic, at zero (T0) and 60 minutes after (T60). GPx 5U and cysteine 0.5mM increased (P<0.05) IAc at T0, when compared to T60. Cysteine 1mM resulted in a higher (P<0.05) IAc on T60, than GPx 1 and 5U, and cysteine 0.5mM. The PMM from a stallion on T60 was higher (P<0.05) than those of two stallions. In sperm kinematic, VCL and VAP were higher (P<0.05) at T0 compared to T60 for the control group, and one stallion showed larger (P<0.05) kinematic values than other animals. It is concluded that the addition of glutathione peroxidase at concentrations 1U and 5U, and cysteine, at concentrations of 0.5mM and 1mM, does not interfere with the integrity of cryopreserved equine sperm, but preserves the kinetic parameters VCL and VAP after 60 minutes of incubation. It should be noted also that the stallion has a strong influence on sperm characteristics post-freezing.

High resveratrol or quercetin concentrations reduce the oscillation index of frozen goat semen

O objetivo deste estudo foi avaliar o efeito de diferentes concentracoes de transresveratrol ou quercetina sobre a capacidade dos espermatozoides caprinos de resistirem a congelacao. Seis pools de semen, obtidos de seis reprodutores caprinos, foram processados com diferentes concentracoes de resveratrol ou quercetina (Experimento 1: 0, 15, 25, 50, 75 ou 100μM de resveratrol; Experimento 2: 0, 15, 25, 50, 75 ou 100μM de quercetina) e congelados. Apos o descongelamento, o semen foi avaliado quanto a cinetica espermatica, a integridade das membranas plasmatica e acrossomal, a morfologia e ao estresse oxidativo nos tempos zero e uma hora de incubacao. Imediatamente apos a descongelacao (zero hora), o wobble (indice de oscilacao) nos grupos tratados com 100μM de quercetina ou de resveratrol foi menor (P<0,05) do que nos tratados com 0 e 25μM de resveratrol e com 0μM de quercetina, respectivamente. Apos uma hora de incubacao, a motilidade total dos tratamentos com 15, 50 e 75µM de quercetina, assim como a integridade de membrana plasmatica em todas as concentracoes de quercetina, foi menor (P<0,05) do que a zero hora. Em oposicao, a linearidade das amostras de semen tratadas com 100µM de quercetina e a retilinearidade daquelas tratadas com 75µM e 100µM de quercetina foram menores (P<0,05) a zero hora do que a uma hora apos descongelacao. Assim, pode-se concluir que o resveratrol e a quercetina, em concentracoes elevadas (100μM), reduzem, transitoriamente, o indice de oscilacao de espermatozoides caprinos submetidos a congelacao e que a quercetina (75 e 100µM) aumenta a linearidade e a retilinearidade ao longo do tempo, o que pode ser favoravel a fertilidade.

Cryopreservation of ram epididymis spermatozoa post-slaughter - A feasible biotechnique?

Background Gametes for breeding high-value livestock is commonly preserved in gene banks for later use in assisted reproduction programs. Such material can be used in artificial insemination or embryos transfer in the field as well as embryos in vitro production [1]. However, when these animals die, genetic material may be lost if it is not possible to recover and preserve spermatozoa from epididymis. Thus, our aim is to establish a recovery technique of ram sperm epididymal post-slaughter and to test its postcryopreservation viability.