Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen supplemented with catalase or Trolox

Semen manipulation and cryopreservation-thaw procedures may accelerate the generation of reactive oxygen species (ROS). Sperm exposure to large amounts of ROS has been shown to cause membrane lipid peroxidation and cellular injury to the sperm. The objective of this study was to overcome the ROS production in frozen-thawed ram semen by the addition of the antioxidants catalase or Trolox to semen following thawing. Frozen-thawed ram semen (100 x 10(6) sperm/straw) was supplemented with PBS (control group), 100 mu g/ml catalase, or 100 mu M Trolox/10(8) sperm (catalase and Trolox being dissolved in PBS) and incubated (37 degrees C) for 5 min. Under the experimental conditions used in this study, the catalase and Trolox antioxidants failed to protect the sperm from the spontaneous production of ROS. However, when lipid peroxidation was induced by iron (FeSO(4)), the addition of Trolox promoted a reduction (P < 0.05) in the formation of TBARS in the semen, compared to the control and catalase semen samples. The generation of TBARS and H(2)O(2) occurred in the extender alone, without the presence of sperm cells. In conclusion, the addition of Trolox to frozen-thawed ram semen could be beneficial as it decreases the production of TBARS when oxidative stress is induced. It is possible that a longer incubation period could lead to different results. The concentration of catalase also needs to be further evaluated. The extender could contribute to the oxidative stress of sperm, as it is a source of ROS during the cryopreservation of semen. (C) 2010 Elsevier B.V. All rights reserved.

Freezing of stallion epididymal sperm

Inseminations with frozen-thawed epididymal sperm have resulted in low-pregnancy rates of mares. If fertility of epididymal sperm could be improved, it would help to preserve genetic material from stallions that have suffered severe injuries, been castrated or have died. The aim of the present study was to investigate the effect of different extenders and pre-freezing addition of capacitation media on freezability of epididymal sperm and on storage at 5 degrees C for 24 h. In experiment 1, epididymal sperm samples were diluted and subsequently frozen with three different extenders: Botu-Crio((R)), EDTA-Lactose and INRA-82. Motility analysis using computer assisted sperm analyzer (CASA) demonstrated better motility for sperm in Botu-Crio((R)) than in the other extenders; EDTA-Lactose yielded better motility than INRA-82 on most evaluated parameters. There was no difference in membrane integrity among the studied extenders. From 18 inseminated mares, 12 (66%) were pregnant 15 days after AI with frozen-thawed epididymal sperm showing that Botu-Crio((R)) was able to maintain the fertility potential. In experiment 2, the effect of incubation of epididymal sperm before freezing in three capacitation media (Fert Talp, Sperm Talp, Talp + Progesterone), seminal plasma, or control was tested. Based on post-thaw motility evaluation by CASA, samples incubated in Sperm Talp showed better motility values. There were no differences in plasma or acrosomal membranes or in mitochondrial potential among groups. We concluded that Botu-Crio((R)) was better than the other extenders in the ability to preserve epididymal sperm and that pre-freeze addition of Sperm Talp was also beneficial. (c) 2008 Published by Elsevier B.V.

Methods of Concentrating Stallion Semen

Removal of excess seminal plasma is sometimes necessary to increase the quality and the longevity of cooled equine semen; moreover, this procedure is an indispensable step aiming to concentrate the sperm cells before freezing equine semen. Typically, the removal of seminal plasma is achieved by centrifugation; however, studies have shown that the force and duration of centrifugation can damage sperm cells and reduce the sperm recovery rate. Recently, new methodologies, such as cushion and filtration, have been described that aim to decrease the mechanical damage of centrifugation to sperm cells. This study aims to compare different methods for concentrating stallion semen.

Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen cryopreserved in extenders with antioxidants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Advances in stallion's epididymal sperm technology

Freezing epididymal sperm is a method to preserve germplasm from animals with not only high genetic potential but also endangered species. In the equine some owners have chosen this possibility in cases of either severe illness or death of stallions. However, the lack of knowledge and poor published results of such technique hampers its propagation. New procedures have allowed some improvement on fertility rates of frozen sperm from the epididymis of stallions. The aim of this study is to report the advances on processing and cryopreservation of samples from the stallion's epididymal semen.

Effect of refrigeration systems upon frozen bull sperm viability assessed by computer-assisted sperm analysis and fluorescent probes

Sperm cryopreservation success depends upon the maintenance of spermatozoa fertility potential. Sperm cells must preserve both integrity and functionality of several cell structures. The stabilization phase must allow the exit of water from the sperm cells via osmosis. This study aimed to compare the effect of refrigeration in the commercial refrigerator (CR) and the transport/refrigeration box (TRB) upon the viability of frozen bull sperm diluted in three different extenders (A, B and C). Ten Nellore bulls, Bos taurus indicus maintained in Artificial Insemination Center were used and the spermatozoa samples was assessed for Plasma Membrane Integrity and CASA evaluation. The stabilization phase (5 degrees C/4 hours) was performed in the CR as well as in the TRB, and then samples were exposed to nitrogen vapor during 20 minutes and then plunged into nitrogen. The statistical analysis was done using the variance analysis and the significance level was set at 5%. In the CR the post-thawing parameters for PM and ALH were higher (p < 0.05) in the extender A (glicine egg-yolk) and extender B (glicine egg-free) when compared with extender C (TRIS egg-yolk). As for BCF, STR and LIN, the parameters were higher (p < 0.05) in extender B than in C. Samples that were stabilized in the TRB presented higher post-thawing parameters (p < 0.05) for PM and LIN in extender A and extender B when compared with C. BCF and STR parameters were higher (p < 0.05) in extemder B when compared with C. Extender B samples had higher (p < 0.05) PMI when stabilized in CR. The findings in this experiment enable us to say that both CR and TRB were effective in keeping the viability of post-thawing bull semen.

Detection of Leptospira spp. from pure cultures and from experimentally contaminated bovine semen by polymerase chain reaction

Considerando a importância do sêmen na transmissão da leptospira bovina, foi realizado o presente estudo que teve como objetivo aplicar a reação em cadeia pela polimerase (PCR) para a detecção de leptospiras em sêmen bovino experimentalmente contaminado. A reação de PCR foi capaz de amplificar um fragmento de DNA específico de 330 pares de bases a partir de cultivos puros de 26 sorovares de Leptospira spp. A contaminação experimental de sêmen com Leptospira interrogans serovar hardjo revelou que a técnica de PCR conseguiu detectar 10 bactérias/ml, concentração sensivelmente mais baixa que as 1.000 bactérias/ml detectadas através do cultivo microbiológico. Os resultados observados revelam o grande potencial da reação de PCR para a detecção de Leptospira spp. em sêmen bovino, notadamente em centrais de inseminação artificial.

The Effect of Season on Semen Characteristics and Freezability in Bos indicus and Bos taurus Bulls in the Southeastern Region of Brazil

The objectives of this study were to evaluate the effects of season in southeast of Brazil comparing genotypes on semen characteristics, freezability and peripheral plasma concentrations of testosterone. Ejaculates of five Bos indicus bulls and six Bos taurus bulls were evaluated over a period of 27 months, which was divided into winter (July, August, September), spring (October, November, December), summer (January, February, March) and autumn (April, May, June). Semen was evaluated according to standard procedures for ejaculate volume, sperm concentration, gross-motility, progressive motility and sperm morphology. After preparing and freezing the ejaculates according to commercial procedures, the straws were stored in liquid N(2) until post-thaw evaluation. Ejaculate volume, sperm concentration, gross-motility, progressive sperm motility, vigor and morphological sperm defects were significantly influenced by season and genotype (p < 0.05). Heat tolerance was better in B. indicus bulls than in B. taurus bulls characterized by lower values of sperm abnormalities throughout the observation period. The highest values were recorded for abnormal heads followed by cytoplasmatic droplets in B. taurus bulls. The proportion of ejaculates which were eliminated before freezing for reasons of bad quality was lower in the B. indicus bulls. Temporal changes in peripheral plasma testosterone concentrations were higher in B. indicus bulls than in B. taurus bulls not revealing seasonal influences. The results of this study show clear genotype differences regarding semen quality. Freezability of B. taurus semen varies considerably throughout the year, leading to a high proportion of eliminated ejaculates. Collecting semen from B. taurus bulls during the summer in an artificial insemination centre may not be profitable.