Morphological and functional characteristics of chilled semen obtained from domestic feline epididymides (Felis catus)

The objective of this study was to determine morphological and functional characteristics of semen retrieved from the feline epididymis before and after cooling. Sixteen adult male cats were orchiectomized. The distal portion of the epididymis and proximal part of the deferent ducts were dissected and squeezed to obtain their content. After centrifugation, the supernatant was removed, sperm were resuspended in a 0.9 mL Tris-fructose-citric acid extender containing 20% egg yolk, aliquoted into three 0.3 mL samples, placed in a refrigerator (4.8 degrees C) and cooled (0.5 degrees C/min). Semen evaluations were performed on four occassions: immediately after epididymal sperm retrieval (TO), and at 24 h (T-1), 48 h (T-2) and 72 h (T-3) after cooling. on each occasion, progressive motility, vigor and sperm morphology were determined. Mean motility and vigor decreased (P < 0.05) between each successive examination. Although the majority of sperm cell damage occurred within the first 24 h, there was a decrease (P < 0.05) in mean percentage of morphologically normal sperm between To and each evaluated time (T-1, T-2, T-3) after cooling, due to an increase in coiled and bent sperm tails. Further studies are needed to evaluate the effects of cooling on the fertilizing capacity of cat epididymal spermatozoa in assisted reproduction programs. (c) 2006 Elsevier B.V. All rights reserved.

Influence of semen storage and cryoprotectant on post-thaw viability and fertility of stallion spermatozoa

The aim of the current study was to verify that stallion, spermatoza could be cooled for 24 hours and then frozen. In experiment I, one ejaculate from each of 13 stallions was used. Semen was collected and split into two parts; one part immediately frozen using standard cryo-preservation techniques and the other diluted, stored in an Equitainer for 24 hours, and then frozen. In experiment II, one ejaculate from each of 12 stallions was collected, diluted with Botu-Semen, and split into two parts: one cooled in an Equitainer and the other in Max-Semen Express without prior centrifugation. After 24 hours of cooling, the samples were centrifuged to remove seminal plasma and concentrate the sperm, and resuspended in Botu-Crio (R) extender containing on e of three cryoprotectant treatments (1% glycerol + 4% dimethylformamide, 1% glycerol + 4% dimethylacetamide and 1% glycerol + 4% methylformamide), maintained at 5 degrees C for 20 minutes, then frozen in nitrogen vapour. No difference was observed between the two cooling systems. The association of 1% glycerol and 4% methylformamide provided the best post-thaw progressive motility. For experiment III, two stallions were used for a fertility trial. Forty three inseminations were performed using 22 mares. No differences were seen in semen parameters and pregnancy rates when comparing the two freezing protocols (conventional and cooled/frozen). Pregnancy rates for conventional and cooled/frozen semen were, respectively, 72.7% and 82.3% (stallion A), and 40.0% and 50.0% (stallion B). We concluded that cooling equine-semen for 24 hours before freezing while maintaining sperm viability and fertility is possible.

Characteristics of seminal plasma proteins and their correlation with canine semen analysis

The objectives were to separate canine seminal plasma proteins (with SDS-PAGE) and to determine the correlation between specific proteins and semen characteristics. Three ejaculates from 20 mixed-breed dogs, of unknown fertility, were collected by digital manipulation. Ejaculate volume and color, sperm motility, sperm vigor, percentage of morphologically normal spermatozoa, and membrane integrity (hypoosmotic swelling test and fluorescent staining) were assessed. For each dog, seminal plasma was pooled from all three ejaculates and proteins were separated with SDS-PAGE, using polyacrylamide concentrations of 13% and 22% in the separation gels. After staining, gel images were digitized to estimate molecular weights (MW) and integrated optical density (IOD) of each lane and of individual bands. Total seminal plasma protein concentration was 2.19 +/- 1.56 g/dL (mean +/- SD; range 1.12-5.19 g/dL). A total of 37 protein bands were identified (although no dog had all 37 bands). In the 13% gel, molecular weights ranged from 100.6 to 17.1 kDa, with four bands (49.7, 33.2, 26.4, and 19.5 kDa) present in samples from all dogs. In the 22% gel, molecular weights ranged from 15.6 to 3.6 kDa, with nine bands (15.6, 13.5, 12.7, 11.7, 10.5, 8.7, 7.8, 5.6, and 4.9 kDa) present in samples from all dogs. Combined for both gels, the majority of bands (85%) had molecular weights < 17 kDa, with B20 (15.6 kDa) in high concentrations in samples from all dogs. There were positive correlations (P <= 0.01) between two bands, 134 (67 kDa) and B5 (58.6 kDa), and sperm motility (r = 0.66 and r = 0.46), sperm vigor (r = 0.56 and r = 0.66), percentage of morphologically normal spermatozoa (r = 0.55 and r = 0.59), the hypoosmotic swelling test (r = 0.76 and r 0.68), and fluorescent staining (r = 0.56 and r = 0.59), respectively. In conclusion, 37 proteins were identified in seminal plasma; two were significantly correlated with semen characteristics. (c) 2007 Elsevier B.V. All rights reserved.

Acrosomal ultrastructure of stallion spermatozoa cryopreserved with ethylene glycol using two packaging systems

The present experiments aimed to examine the substitution of glycerol (G) by ethylene glycol (E) as a cryoprotective agent for stallion spermatozoa. Two different ethylene glycol concentrations (5% and 10%) and also the association of glycerol (2%) and ethylene glycol (3%) (E/G) were studied (Experiment 1). In Experiment 2, two packing systems (0.5 x 4.0 ml) were evaluated using both cryoprotectors. In both experiments, the sperm membrane integrity after freezing was evaluated using transmission electron microscopy. The mean post-thaw motility was 34.25, 36.5, 29.25 and 34.75% for G5%, E5%, E10% and E/G, respectively. It was observed that the percentage of motile spermatozoa was significantly smaller (P<0.05) when semen was processed with E10%. A decrease in the acrosome integrity was observed in frozen thawed spermatozoa from all treated groups. It was observed that 28.0, 22.5, 25.5 and 22.5 % of the sperm cells had a normal acrosome following freezing with G5%, E5%, E10% and E/G, respectively. Undulation of the outer acrosomal membrane, acrosomal swelling and loss of acrosomal content density and homogeneity were the most evident ultrastructural alterations observed. In Experiment 2, the post-thaw motility was higher (P<0.05) for sperm frozen in 0.5 ml straws than in 4.0 mi straws, regardless of the cryoprotector used. The ultrastructural evaluation showed 26.7 and 16.0% of intact acrosomes for sperm frozen in 0.5 ml and 4.0 ml straws, respectively. We concluded that ethylene glycol has similar cryoprotective properties to glycerol and that utilisation of 0.5 ml straws improved the ability of horse sperm cells to withstand damage after the cryopreservation process.

Amides as cryoprotectants for freezing stallion semen: A review

Stallion semen cryopreservation, despite its impact on the horse industry, is not an established technology. During the last years, a number of modifications have been proposed to the freezing process, however, a large population of stallions still have poor semen quality and fertility after frozen-thawed. Glycerol toxicity could be a reason for the variation on stallion sperm freezability. There are limited publications concerning the use of alternative cryoprotectants for equine sperm. Glycerol is contraceptive for some species and other cryoprotectors, such as amides, have been show to be a good option for freezing semen of these species. Recent reports have shown encouraging data respecting the use of amides as cryoprotectants for stallions, with more remarkable improvements for semen from stallions that freeze poorly when glycerol is used. (C) 2005 Elsevier B.V. All rights reserved.

Vasectomy effect on canine seminal plasma biochemical components and their correlation with seminal parameters

Three semen samples were collected at 48 It intervals from 20 mature research dogs previously conditioned to manual semen collection. Vasectomy was performed in all dogs, and 15 days after surgery, another three ejaculates were similarly collected. The semen was evaluated, and centrifuged to obtain seminal plasma for measurement of pH, and concentrations of total proteins (TP), total chlorides (Cl), calcium (Ca), potassium (K), and sodium (Na). The seminal plasma protein profile was evaluated by SDS-PAGE; molecular weights and the integrated optical density (IOD) of each band were estimated. There was a negative correlation between K concentration and progressive motility (r = -0.49, P = 0.027), sperm vigor (r = -0.60, P = 0.0053), and plasma integrity, evaluated by both the hypo-osmotic swelling test (r = -0.50, P = 0.026) and a fluorescent stain (r = -0.45, P = 0.046). Positive correlations between Na and K pre- and post-vasectomy (r = 0.88, P < 0.001; r = 0.56, P < 0.01, respectively) were verified. There were a total of 37 bands pre-vasectomy and 35 post-vasectomy (range, 100.6-3.6 kDa). Bands B9 and B13 (42.6 and 29.2 kDa) were not present post-vasectomy. The IOD of band B3 (73.5 kDa) was higher (P 0.03) pre-vasectomy, compared to post-vasectomy; conversely, the IODs of bands B29 and B37 (7.8 and 3.6 kDa) increased (P 0.026 and 0.047). Pre-vasectomy, there was a positive correlation (r = 0.49, P = 0.029) between band B37 band (3.6 kDa) and the Na:K ratio. In conclusion, K appeared to be involved in sperm motility in dogs and could be a tool to evaluate sperm function. The prostate contributed several elements to canine seminal plasma. Vasectomy changed Ca concentrations and the protein profile of the seminal plasma. Further studies must be performed to clarify the function of these elements on the in vivo fertility of dogs. (c) 2006 Elsevier B.V. All rights reserved.

Heparin-binding proteins of canine seminal plasma

Heparin-binding proteins (HBP) from seminal plasma have been expected to participate in modulation of the acrosomal reaction, and have been correlated with fertility in some species. However, they have not been described in the dog. The aim of this study was to document the HBPs of canine seminal plasma. Six pooled samples of seminal plasma from three crossbred dogs were used. The HBPs were isolated by heparin affinity chromatography and the fractions recovered were pooled. One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on 12 and 18% vertical minigels. The stained gels were scanned and the molecular weight (kDa) values for each band within a lane were calculated by image analysis software. The electrophoresis analysis of the pooled eluded fractions identified 19 bands, with molecular weights varying from 61.5 to 5.2 kDa. Previous studies, using one-dimensional SDS-PAGE, identified two bands (67 and 58.6 kDa), which were positively correlated with some semen parameters (sperm motility, sperm vigor, percentage of morphologically normal sperm and plasma membrane integrity). The 61.5 kDa band detected in the present study apparently corresponded to the 58.6 kDa band identified previously. Canine seminal plasma contained HBP; since HBP modulate the acrosome reaction in other species, they may have the same function in the dog. Further studies are necessary to better characterize this protein and determine if it is associated with fertility in the dog. (c) 2006 Elsevier B.V. All rights reserved.

Evaluation testicular fine needle aspiration cytology and serum testosterone levels in dogs

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

Semen cryopreservation protocols of Mangalarga Marchador stallions

The effect of the utilization of three semen protocols (Inra 82®, Merck Gema and Botu-crio®) and two filling techniques (0.25 and 0.50 mL straws) in Mangalarga Marchador stallions were studied in this experiment. Sperm parameters were assessed during processing and post-freezing. No interactions between the protocols and type of filling were observed, so they were assessed separately. Sperm parameters were not altered when the extender was added to the centrifugation; however, there was reduction of motility and strength when freezing extenders were added. The Botu-crio® protocol preserved the parameters of total and progressive sperm motility, smoothed path velocity (µm/s), straight line velocity (µm/s), track velocity (µm/s) and the average and fast spermatozoa percentage better than the others. No difference between the extenders for the percentage of sperm integrity was observed. There was no difference in the responses studied on the filling techniques. The stallions presented better freezing with the use of the Botu-crio® protocol. The best post-freezing viability results were found for semen frozen using the Botu-crio® protocol and there were no differences concerning the sperm quality comparing 0.25 and 0.50 mL straws.

Comparison of in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or new lecithin based extenders

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

Comparison of efficiency between two artificial insemination methods using frozen-thawed semen in domestic cat (Felis catus) Artificial insemination in domestic cats

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

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.

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)

PCR Multiplex fluorescente para detecção de bactérias em sêmen bovino

Este estudo teve como objetivo avaliar o limiar de detecção da técnica de PCR multiplex fluorescente aliada a eletroforese capilar na detecção de agentes infecciosos em amostras de sêmen experimentalmente contaminadas com concentrações decrescentes das bactérias Brucella abortus, Leptospira interrogans sorovar pomona, Campylobacter fetus e Haemophilus somnus. Amostras de sêmen bovino foram experimentalmente contaminadas com concentrações decrescentes de bactérias obtidas através de diluições seriadas na base 10 de modo a obter-se amostras contendo desde 1 vez até 10-7 bactérias/mL a partir da concentração inicial de Leptospira pomona, Brucella abortus, Campylobacter fetus e Haemophilus somnus. As diluições foram efetuadas individualmente para cada bactéria, bem como nas diferentes concentrações necessárias para a padronização do teste de multiplex PCR. As extrações de DNA de todas as soluções contendo espermatozóides e bactérias analisadas no presente estudo foram realizadas segundo protocolo descrito por Heinemann et al. (2000). Os produtos de PCR multiplex foram avaliados por eletroforese em gel de poliacrilamida 8% e separação eletroforética por sistema capilar em equipamento automático de análise de fragmentos de DNA MegaBace. Observou-se a amplificação de fragmentos de 193pb, 330pb, 400pb e 415pb a partir do DNA de B. abortus, L. pomona, H. somnus, C. fetus, respectivamente. Na análise por eletroforese capilar de produtos da PCR multiplex do DNA para detecção simultânea dos quatro patógenos observou-se a sinal de positividade até a diluição de 10-3 bactérias/mL vezes da concentração inicial da solução estoque de cada bactéria. A técnica de PCR multiplex aliada à eletroforese capilar foi usada pela primeira vez para o diagnóstico direto de quatro bactérias patogênicas no sêmen, demonstrando ser um método rápido na detecção de bactérias causadoras de doenças reprodutivas.