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.

Thermoresistance sperm tests are not predictive of potential fertility for cryopreserved bull semen

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

Viability of primordial follicles derived from cryopreserved ovine ovarian cortex tissue

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

Viability of OPS Vitrified Sheep Embryos After Direct Transfer

The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions.

Motility and viability of ram sperm cryopreserved in a Tris-egg yolk extender supplemented with anti-oxidants

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.

Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Histochemical and morphological characteristics of the levator ani muscle in rats

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

Comparison between RT-PCR and the mouse inoculation test for detection of rabies virus in samples kept for long periods under different conditions

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

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)

Growth hormone attenuates skeletal muscle changes in experimental chronic heart failure

Objective: This study evaluated the effects of growth hormone (GH) on morphology and myogenic regulatory factors (MRF) gene expression in skeletal muscle of rats with ascending aortic stenosis (AAS) induced chronic heart failure.Design: Male 90-100 g Wistar rats were subjected to thoracotomy. AAS was created by placing a stainless-steel clip on the ascending aorta. Twenty five weeks after surgery, rats were treated with daily subcutaneous injections of recombinant human GH (2 mg/kg/day; AAS-GH group) or saline (AAS group) for 14 days. Sham-operated animals served as controls. Left ventricular (LV) function was assessed before and after treatment. IGF-1 serum levels were measured by ELISA. After anesthesia, soleus muscle was frozen in liquid nitrogen. Histological sections were stained with HE and picrosirius red to calculate muscle fiber cross-sectional area and collagen fractional area, respectively. MRF myogenin and MyoD expression was analyzed by reverse transcription PCR.Results: Body weight was similar between groups. AAS and AAS-GH groups presented dilated left atrium, left ventricular (LV) hypertrophy (LV mass index: Control 1.90 +/- 0.15; AAS 3.11 +/- 0.44; AAS-GH 2.94 +/- 0.47 g/kg; p < 0.05 AAS and AAS-GH vs. Control), and reduced LV posterior wall shortening velocity. Soleus muscle fiber area was significantly lower in AAS than in Control and AAS-GH groups; there was no difference between AAS-GH and Control groups. Collagen fractional area was significantly higher in MS than Control; AAS-GH did not differ from both Control and AAS groups. Serum IGF-1 levels decreased in AAS compared to Control. MyoD mRNA was significantly higher in AAS-GH than AAS; there was no difference between AAS-GH and Control groups. Myogenin mRNA levels were similar between groups.Conclusion: In rats with aortic stenosis-induced heart failure, growth hormone administration increases MyoD gene expression above non-treated animal levels, preserves muscular trophism and attenuates interstitial fibrosis. These results suggest that growth hormone may have a potential role as an adjuvant therapy for chronic heart failure. (C) 2009 Elsevier Ltd. All rights reserved.

Avaliações sensoriais, bioquímicas e microbiológicas do guacamole, um produto à base de abacate, sob armazenamento a frio e com adição de ácido ascórbico

The elaboration of avocado products for commercialization keeping their characteristics of fresh product has been limited. The cut avocado darkens quickly and their sensorial characteristics are modified with the storage. In the present research, the sensorial parameters, microbiological stability, and peroxidase and polyphenoloxidase activity were evaluated in guacamole added with ascorbic acid and conserved under low temperature, by using avocado variety Hass. Products were conditioned in polyethylene+nylon packages with and without vacuum application; then, they were subjected to the slow and fast freezing (-18 degrees C) and stored in freezer (-18 degrees C). Evaluations were performed at the moment of elaboration of the product (t0) and at 3, 7 and 30 days post-storage. At t30, samples were kept under refrigeration (4 +/- 1 degrees C) and evaluated at 3, 5 and 7 days. After the 30 days of storage, -18 degrees C under freezing, followed by thawing and keeping at 4 +/- 1 degrees C for 7 days, the notes for the sensorial parameters decreased. The peroxidase activity was totally inhibited in the elaborated product and the polifenol oxidase activity considerably decreased in the guacamole (20.07 mM catechol/g fresh matter) relative to those in the fruit (58.31 mM catechol/g fresh matter), however with no significant variation during storage (at -18 degrees C). The samples were microbiologically stable under the conditions of the present study. The addition of ascorbic acid contributed to the conservation of the frozen avocado product by decreasing the enzymatic activity. However, the sensorial parameters are prejudiced under thawing and storage at 4 +/- 1 degrees C.

Quantitative distribution of gibberellins and indole-acetic acid in pea (Pisum sativum L.) seedlings

The objective was to determine the distribution in pea seedling of GA (by thin layer chromatography) and IAA (by HPLC). Three samples of 30 7/day-old seedlings were sectioned into: apical meristem + plumular hook; stem elongation zone; stem non-elongation zone; cotyledons; root non-elongation zone; root tip. The material was frozen in liquid N and stored at -80 degrees C. The stem elongation zone, the apical meristem+plumular hook and the root tip had low IAA, whereas the stem non-elongation zone and root non-elongation zone had high IAA content. The stem elongation zone and apical meristem plumular hook had high GA, while root non-elongation zone had the lowest GA content.

Efeito do cobre na atividade da enzima pirogalol peroxidase em plantas de Myriophyllum aquaticum cultivadas em solução nutritiva

Myriophyllum aquaticum é uma planta perene, herbácea, que pode se desenvolver totalmente submersa ou com a porção terminal dos ramos acima da superfície da água. É também considerada uma planta daninha que possui elevado potencial de colonização, o qual, dependendo da densidade populacional, pode causar aumento no teor de matéria orgânica e redução de oxigênio na água, comprometendo a qualidade da água e seus usos múltiplos. O objetivo do presente trabalho foi verificar a influência do cobre na atividade da pirogalol peroxidase de plantas de M. aquaticum submetidas à solução nutritiva contendo concentrações de cobre de 1,2; 11,2; 21,2; 31,2; e 41,2 µg L-1. O experimento foi conduzido em um delineamento experimental inteiramente casualizado, com quatro repetições e cinco tratamentos, aos quais as plantas foram submetidas durante 21 dias. Aos 81 dias após a instalação das mudas em solução nutritiva contendo os diferentes níveis de cobre, as folhas foram colhidas a partir do ápice da planta até o final do ramo, que não estavam em contato com a solução. Esse material fresco foi envolvido por plástico transparente e papel-alumínio e, a seguir, congelado em nitrogênio líquido, sendo armazenado em freezer a -20 ºC até o momento da determinação da atividade da enzima pirogalol peroxidase. A atividade da enzima foi progressiva com o aumento das doses de cobre. As plantas cultivadas com 40 µg L-1 de Cu2+ após três semanas, com base em avaliação visual, apresentaram redução no desenvolvimento.

Ischaemia and reperfusion effects on skeletal muscle tissue: morphological and histochemical studies

This was a study on the oxidative stress due to ischaemia (I) and reperfusion (R) in skeletal muscle tissue. Using a tourniquet, groups of rats were submitted to ischaemia for 4 h, followed by different reperfusion periods. The animals were divided in four groups: control; 4 h of ischaemia (IR); 4 h of ischaemia plus 1 h reperfusion (IR-1 h); 4 h of ischaemia plus 24 h reperfusion (IR-24 h); and 4 h of ischaemia plus 72 h reperfusion (IR-72 h). At the end of the procedures, samples of soleus muscle were collected and frozen in n-hexane at -70 degrees C. Cryostat sections were submitted to haematoxylin-eosin, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) stains. An additional muscle sample was processed for electron microscopy. No alterations were found in control animals. IR group showed fibres had normal aspect besides some round, acidophilic and hypertrophic fibres. There were several fibres with angular outlines and smaller diameters in this group compared with control group. NADH-TR/SDH reaction was moderately intense in most fibres. In some fibres, cytoplasm showed areas without activity and other fibres had very intense reactivity. IR-1 h group showed oedema hypercontracted fibres with disorganized myofibrils, mitochondria with focal lesions and dilated sarcoplasmic reticulum. NADH-TR/SDH reaction was moderate to weak. IR-24 h showed intense inflammatory infiltrate in the endomysium and perimysium. NADH-TR/SDH reaction was similar to IR-1 h. IR-72 h showed necrotic fibres, areas with inflammatory infiltrate, reduced muscle fibres at different stages of necrosis and phagocytosis, and many small round and basophilic fibres characterizing a regeneration process. NADH-TR/SDH reaction was weak to negative. Our results suggest that ischaemia and the subsequent 1-, 24- and 72-h reperfusions induced progressive histological damage. Although progressive, it may be reversible because there were ultrastructural signs of recovery after 72-h reperfusion. This recovery could in part be due to the low oxidative stress identified by the morphological and histochemical analysis.