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.

Impact of proximal cytoplasmic droplets on quality traits and in-vitro embryo production efficiency of cryopreserved bull spermatozoa

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

Qualidade espermática de sêmen de cães naturalmente infectados por Leishmania sp:

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

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.

A histological study of the seminal vesicle of the armoured catfish Corydoras aeneus

Most species of Corydoras exhibited a reproductive behaviour called 'T-position', and exhibited an accessory gland in the male genital tract, called the seminal vesicle. It appeared that both the structure and the composition of the fluid varied considerably between the species investigated. Consequently, different opinions were proposed regarding the possible role of seminal vesicle on this particular reproductive behaviour. Male adults of Corydoras aeneus were collected, anaesthetized, and samples of seminal vesicle were fixed in Bouin's solution. The sections were stained with haematoxylin-eosin and periodic acid Schiff. The seminal vesicle showed a system of anastomosed secretory tubules, forming a vesicular collective network, which gave rise to the vesicular ducts. The latter fused with the testicular efferent ducts and formed the spermatic ducts. Considering this fusion, when the sperm cells reached the spermatic ducts, the fluid produced at the seminal vesicle covered them. Histochemical studies evidenced the presence of neutral and acid glycosaminoglycans in the seminal fluid. Considering the reproductive behaviour of C. aeneus, it is believed that the protection associated with the immobilization of the sperm cells assures the sperm integrity during the passage through female's intestine until fertilization.

Morphological aspects of spermatogenic cells at different stages of sexual maturation in black isogenic C57BL/6/Uni mice

In order to study the morphological changes that occur in cells of the testes of isogenic black mouse C57BL/6/Uni into three periods during spermatogenetic used 15 mice divided into 3 groups of 5 animals with 40,50 and 60 days of age. The mice were sacrificed and weighed. Testicles were weighed and measured, and histologically processed and stained with HE, PAS and Masson Massom-H and evaluated under light microscopy. It was observed that group I with 40 days of age in the seminifcrous tubules had a lumen with sparse small amount of interstitial tubular cells. In the seminiferous epithelium type A spermatogonia, intermediate and B were identified, which occupied the compartment adbasal and intermingled with these cells in spermatocytes I in Pachytene and leptotene was observed, whereas in the adluminal compartment Golgi phase spermatids we observed the presence of acrosomal granule. In group II, the cells of the seminiferous epithelium were developed and it was observed in round spermatids cephalic hood phase plus many elongated spermatids in acrosome phase and Sertoli cells. In Group III, 60 days old, it was found that seminiferous epithelium which was of the tubules had elongated spermatids in acrosome phase and maturation, with elongated nuclei and acrosomal system typical of spermiation in the presence of sperm and residual bodies near the tubular lumen. Therefore morphological evolution of germ cell testicular spermatids can be checked and recognized in its four phases: Golgi, cap, acrosome and maturation over the age of the animal.

Ultrastructure of the epithelium lining of cauda epididymidis in mongrel dogs

The epithelium lining of cauda epididymidis in mongrel dogs was examined by transmission electron microscopy. The epididymal epithelium is pseudostratified with stereocilia and is composed predominantly of principal and clear cells. Therefore, exist basal and apical cells. The principal and clear cells show features suggesting that they may be preferentially involved in absorptive and secretive functions. These results are compared with previously published data on the cauda epididymidis of other mammalian species, in order to understand the significance of the epididymis in sperm maturation.

Influence of Steroidal Anti-Inflammatory Drugs on Viability and Fertility of Equine Semen

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

Epididymis-specific pathologic disorders in rats exposed to gossypol from weaning through puberty

Previous work in our laboratory revealed that the pubertal period of reproductive development in the male rat was particularly vulnerable to gossypol exposure, with a higher frequency of round structures in the lumen of the cauda epididymidis in the treated rats. Herein, we utilized hemicastration and electron microscopy to confirm that the epididymis is a definitive target of gossypol. Although exposure to gossypol from weaning through puberty caused a significant decrease in daily sperm production, as well as in the concentration of sperm in the epididymis, serum testosterone levels and reproductive organ weights were not altered. In gossypol treated rats, sperm morphology was compromised severely, but the epithelium in testis and epididymis appeared morphologically normal. Ultrastructural examination revealed that round structures, present only in gossypol exposed males, represented: (1) principal cells exfoliated from the epididymal epithelium; (2) epididymal epithelial cell cytoplasm containing degenerating sperm; and (3) degenerating epithelial cells, consisting of vesicles and particles of different sizes, forms and densities. Taken together, the data confirm that gossypol targets the epididymis, disturbing both the structure and function of this organ, and presumably disrupts sperm maturation.

Effects of the hydromethanolic extract of Austroplenckia populnea (Celastraceae) on reproductive parameters of male rats

Austroplenckia populnea (Reiss.) Lundell. was selected for this study because it has been shown that some plants from the Celastraceae family have antifertility effects. Twelve adult male rats were treated with hydromethanolic extract made from the leaves, 500 mg/kg/day, orally, for 70 days. Distilled water was administered to the control animals (n = 10). At the end of the experiment, and before killing the rats, their sexual behavior was evaluated. The number of intromissions, latencies to first mount and ejaculation, and first intromission after ejaculation were significantly reduced in the treated group, but the total number of ejaculations did not differ from the control group. The weight and histology of the reproductive organs, sperm production, spermatogenesis, prostate fructose content, cauda epidydimides duct diameter, and sperm morphology were not affected. Sperm concentration in cauda epidydimides was significantly decreased. The results showed that A. populnea has effects on male rat reproduction, affecting the sexual behavior and epididymal sperm concentration. (C) 2002 Elsevier B.V. All rights reserved.