Association Between DMSO and Sugars in the Sperm Cryopreservation of Pacu.

　　BACKGROUND: The cryopreservation protocol that has been developed exclusively for the preservation of the sperm of the species different. OBJECTIVE: this study was to evaluate the effect of the association of 10% DMSO with trehalose, raffinose, sucrose and lactose concentrations on the sperm cells of Piaractus mesopotamicus. MATERIALS AND METHODS: Sperms were collected from the animals through abdominal massage. The samples were diluted in the Beltsville Thawing Solution without different concentrations of other sugars (test conditions). Sixty days after the cryopreservation, cell movement analysis was performed using CASA. RESULTS: The results revealed that the parameters for total motility and motility period were superior when 100mM raffinose (P <0.05). The lateral displacement of the head was observed to be improved was 100mM lactose, 150mM sucrose and 150mM raffinose (P <0.05) as compared to treatment wherein lactose (0mM) was omitted. CONCLUSION: the results of our study indicated that the ideal parameters for cryopreservation, were obtained when the cryopreservation fluid contained 100mM raffinose in association with DMSO.

Boar sperm quality after supplementation of diets with omega-3 polyunsaturated fatty acids extracted from microalgae.

This study evaluated effects of diet supplementation with omega-3 polyunsaturated fatty acids (PUFA) from microalgae on boar sperm quality. Two groups of boars (n = 3 each) were fed during 75 days either a commercial diet (control), or the same diet supplemented with omega-3 PUFA from the heterotrophic microalgae Schizochytrium sp. (120 g/kg). Sixteen ejaculates were collected per boar. Some sperm kinetics parameters were inferior for supplemented than for control boars (p < .05): distance average path; distance in both curved and straight line; velocity average path, velocity in both curved and straight line; and amplitude of lateral head displacement. Spermatozoa from supplemented boars presented lower mitochondrial functionality, but greater membrane fluidity compared to the control group (p < .01). Membrane and acrosome integrity, production of reactive oxygen species and lipid peroxidation did not differ (p > .05). Serum cholesterol levels were greater (p < .05) for supplemented than for control boars at the 30th and 60th d of supplementation, but levels of triglycerides and IGF-1 did not differ (p > .05). Compared to the control, spermatozoa of supplemented boars were slower, travelled shorter distances and presented impaired energy metabolism, but their greater membrane fluidity may potentially favour their cryopreservation.

Cooling strategies for brazilian flounder Paralichthys orbignyanus embryos.

BACKGROUND: Paralichthys orbignyanus is the species of the greatest potential for marine and estuarine fish farming in southern Brazil. Consequently, embryo cryopreservation becomes an important tool for increasing their production. OBJECTIVE: To evaluate the effects of cooling protocols on the viability of embryos of P. orbignyanus at two stages of development (neurula and early differentiation of the tail). MATERIALS AND METHODS: Control embryos were maintained at 23 degree C and treated embryos were cooled to 15 degree C, 10 degree C and 5 degree C at rapid, moderate and slow cooling rates. Then embryos were maintained at these different temperatures for 30, 60 and 90 min and the loss of viability assessed as hatching rates (HR) and morphologically normal larvae (MNL). RESULTS: The average HR for embryos following cooling was higher for those at the tail stage compared to the neurula stage (P<0.05). In both stages there was no statistical difference between the HR of control embryos and those exposed to rapid cooling. Also for tail stage embryos, there was no difference between MNL of control and rapidly cooled embryos. CONCLUSION: As first steps in the development of cryopreservation methods for P. orbignyanus embryos, the use of a rapid cooling and holding at 5 degree C for 30 min are recommended.

Ovarian fragment sizes affect viability and morphology of preantral follicles during storage at 4°C.

The method of transportation and the conditions imposed on the ovarian tissue are pivotal aspects for the success of ovarian tissue cryopreservation (OTC). The aim of this study was to evaluate the effect of the size of the ovarian tissue (e.g. whole ovary, biopsy size and transplant size) during different times of storage (0, 6, 12 and 24 h) on the structural integrity of equine ovarian tissue transported at 4°C. Eighteen pairs of ovaries from young mares (<10 years old) were harvested in a slaughterhouse and processed to simulate the fragment sizes (biopsy and transplant size groups) or kept intact (whole ovary group) and stored at 4°C for up to 24 h in α-MEM-enriched solution. The effect of the size of the ovarian tissue was observed on the morphology of preantral follicles, stromal cell density, DNA fragmentation and mitochondrial membrane potential. The results showed that (i) biopsy size fragments had more morphologically normal preantral follicles after 24 h of storage at 4°C; (ii) mitochondrial membrane potential was the lowest during each storage time when the whole ovary was used; (iii) DNA fragmentation rate in the ovarian cells of all sizes of fragments increased as storage was prolonged and (iv) transplant size fragments had increased stromal cell density during storage at cool temperature. In conclusion, the biopsy size fragment was the best to preserve follicle morphology for long storage (24 h); however, transportation/storage should be prior determined according to the distance (time of transportation) between patient and reproduction centers/clinics.

Evaluation of sperm quality of Erythrolamprus poecilogyrus sublineatus (Cope, 1860) (Serpentes, Dipsadidae).

Erythrolamprus poecilogyrus sublineatus (Cope, 1860), is a species widely distributed in the Pampa Domain, occurring in Rio Grande do Sul, Argentina and Uruguay, mainlyin the pampa region. In the coastal region of southern Brazil this is serpent is considered one of the most abundant. The purpose of the present study is to describe the techniques of sperm evaluation in vitro for E. poecilogyrus sublineatus in the coastal plain of Rio Grande do Sul, Brazil. After laparatomy the efferent vases were collected and the semen was diluted in 1ml Beltsville Thawing Solution. The characteristics of motility, membrane integrity, mitochondria, acrosome, DNA, cell viability and cellular functionality were evaluated. Fluorescent probes were used for the evaluation of sperm structure in epifluorescence microscope. With the techniques described, it was possible to identify intact and injured cells, enabling the determination of cell characteristics for the spring season (October and November). It was observed in the analyses that 80% of sperm cells were mobile and that 84.1 ± 8.0% of sperm membranes were intact. The standards found were of 48 ± 13.8% of intact acrosome, 73.6 ± 6.0 of perfect DNA and of 91.8 ± 4.0 of functional mitochondria. Thus, these values from the sperm analysis can be used as standards for the species Erythrolamprus poecilogyrus sublineatus.

Quercetin in equine frozen semen.

　　BACKGROUND: Supplementation of sperm diluents to reduce the damage caused by the freeze-thaw cycle is broadly used in equine semen cryopreservation. OBJECTIVE: The present study aimed at determining the most appropriate quercetin supplementation in equine freezing extender. MATERIALS AND METHODS: Quercetin at four different concentrations (0.25, 0.5, 0.75 or 1 mM) was added in the sperm freezing diluent before the freeze-thaw cycle. The spermatozoa population was analyzed by flow cytometry and a statistical analysis was conducted to detect significant differences between control and treated samples. RESULTS: The statistical analysis did not reveal any significant modification of seminal parameters. CONCLUSION: Within the concentrations tested, quercetin supplementation in equine freezing extender did not affect progressive motility, mitochondrial functionality, acrosome reaction, membrane integrity or DNA fragmentation index in post-thaw equine semen.

Effects of cryotherapy combined with therapeutic ultrasound on oxidative stress and tissue damage after musculoskeletal contusion in rats.

OBJECTIVE: To investigate the combined effects of cryotherapy and pulsed ultrasound therapy (PUT) on oxidative stress parameters, tissue damage markers and systemic inflammation after musculoskeletal injury. DESIGN: Experimental animal study. SETTING: Research laboratory. PARTICIPANTS: Seventy male Wistar rats were divided into five groups: control, lesion, cryotherapy, PUT, and cryotherapy+PUT. INTERVENTIONS: The gastrocnemius muscle was injured by mechanical crushing. Cryotherapy was applied immediately after injury (immersion in water at 10°C for 20minutes). PUT was commenced 24hours after injury (1MHz, 0.4W/cm2SPTA, 20% duty cycle, 5minutes). All animals were treated every 8hours for 3 days. MAIN OUTCOME MEASURES: Oxidative stress in muscle was evaluated by concentration of reactive oxygen species (ROS), lipid peroxidation (LPO), anti-oxidant capacity against peroxyl radicals (ACAP) and catalase. Plasma levels of creatine kinase (CK), lactate dehydrogenase (LDH) and C-reactive protein (CRP) were assessed. RESULTS: When applied individually, cryotherapy and PUT reduced CK, LDH, CRP and LPO caused by muscle damage. Cryotherapy+PUT in combination maintained the previous results, caused a reduction in ROS [P=0.005, mean difference -0.9×10-8 relative area, 95% confidence interval (CI) -0.2 to -1.9], and increased ACAP {P=0.007, mean difference 0.34 1/[relative area with/without 2,2-azobis(2-methylpropionamidine)dihydrochloride], 95% CI 0.07 to 0.61} and catalase (P=0.002, mean difference 0.41units/mg protein, 95% CI 0.09 to 0.73) compared with the lesion group. CONCLUSIONS: Cryotherapy+PUT in combination reduced oxidative stress in muscle, contributing to a reduction in adjacent damage and tissue repair.

Migration and reproductive biology of Mugil liza (Teleostei: Mugilidae) in south Brazil.

The mullet Mugil liza occurs along the Atlantic coast of South America from Venezuela to Argentina, but 95% of the commercial catch is collected from south Brazil between São Paulo and Argentina. Mugil liza is a single spawner with oocyte development occurring synchronously in two groups. Spawning happens in marine areas and occurs after migration. The reproductive migration occurs from Argentina (38° S) to the southern Brazilian states (24-26° S) from April to July, with peak spawning in June between northern Santa Catarina and Paraná. The presence of hyaline oocytes was associated with high salinity and sea surface temperatures of 19-21° C, and followed the seasonal northward displacement of these oceanographic conditions. The average size at first maturity (Lm ) for both sexes was 408·3 mm total length, LT . Males (Lm = 400·1) matured earlier than females (Lm = 421·9 mm). Fecundity ranged from 818,992 to 2,869,767 oocytes (mean = 1,624,551) in fish that were between 426 and 660 mm LT .

Influence of mineral, olive or sunflower oils on male reproductive parameters in vitro--the wild rodent Calomys laucha.

The aim of this study was to evaluate the influence of oils on male reproductive parameters in Calomys laucha. Twenty-four animals were distributed into four groups and given the following substances by gavage: water, mineral oil, olive oil and sunflower oil. After 10 days of gavage, the animals were euthanised and the semen was collected from them for assessing acrosome integrity and carrying out in vitro penetration (IVP) test. Acrosome was significantly reduced (P < 0.05) for the vehicles in relation to control. In vitro penetration was reduced in all vehicles in relation to control, but only sunflower oil had statistically lower levels of reduction (P < 0.05). Oily vehicles are able to influence in vitro reproductive tests negatively, interfering in reproductive toxicological studies.

White-tailed deer (Odocoileus virginianus) develop spirochetemia following experimental infection with Borrelia lonestari.

Borrelia lonestari is considered a putative agent of southern tick-associated rash illness (STARI) and is known to occur naturally only in lone star ticks (Amblyomma americanum) and white-tailed deer (Odocoileus virginianus). We used a low passage isolate of B. lonestari (LS-1) to inoculate white-tailed deer, C3H mice, Holstein cattle, and beagles. Animals were monitored via examination of Giemsa and acridine orange stained blood smears, polymerase chain reaction (PCR), indirect fluorescent antibody (IFA) test, and/or culture isolation. Spirochetes were visualized in blood smears of both deer on days post-inoculation (DPI) 6, 8, 12 and one deer on DPI 15. Whole blood collected from deer tested PCR positive starting on DPI 4 and remained positive as long as DPI 28. Both deer developed antibody titers of >64, with a maximum IFA titer of 1024. The organism was reisolated from the blood of both deer on DPI 6 and one deer on DPI 12. All isolation attempts from mice, calves, or dogs were negative, although one of seven mice was transiently PCR positive. Mice and dogs developed an IFA titer > or =64, while calves lacked a detectable antibody response. These preliminary experimental infection trials show that white-tailed deer are susceptible to infection with B. lonestari and develop a spirochetemia following needle-inoculation, while C3H mice, calves, and dogs do not. Results suggest that deer may serve as a vertebrate reservoir host. Tick transmission studies are needed to confirm that this organism can be maintained in a natural cycle involving deer and A. americanum.

Disease agents in Amblyomma americanum from northeastern Georgia.

Amblyomma americanum (lone star tick) is known or suspected to vector several organisms that are implicated as human pathogens, including Ehrlichia chaffeensis, E. ewingii, and Borrelia lonestari. These three agents have also been detected in white-tailed deer (Odocoileus virginianus). Because northeastern Georgia has a high abundance of both lone star ticks and white-tailed deer, and one of these organisms, E. chaffeensis, is already known to be endemic in the area, we assayed individual adult A. americanum, collected during the spring of 2001, 2002, and 2003, for these three organisms. A total of 400 ticks were dissected and tissues assayed by polymerase chain reaction (PCR) using Ehrlichia species-specific and Borrelia genus-wide primers. Of ticks tested, 2.0% (8/398) had evidence of E. chaffeensis, 4.8% (19/398) had evidence of E. ewingii, and 1.0% (4/398) had evidence of B. lonestari. Borrelia sp. spirochetes were also visualized by an indirect fluorescent antibody test, using an anti-flagellin monoclonal antibody (H9724), in a total of 10.7% (32/300) of ticks tested in 2003. These results reconfirm the presence of E. chaffeensis and establish evidence of E. ewingii and B. lonestari in questing adult A. americanum ticks from northeastern Georgia. Detection of at least two of the three organisms in ticks collected each year suggests that people in northeastern Georgia are at risk of infection with these organisms.

Utility of plasmatic levels of alpha-1-antiprotease (A1AP) as a cancer marker.

The objective of this study was to evaluate the diagnostic utility as a cancer marker of plasmatic levels of A1AP. This case-control study included 135 cancer patients from different sites, confirmed histologically, and 95 controls (57 normal individuals plus 38 chronically ill patients with non-tumoral diseases). Determination of A1AP was done by a nephelometric procedure using a laser nephelometer as the measuring instrument. There were no sex or age related variations in plasmatic A1AP. Mean values of A1AP in 57 normal controls was 2.87 milligrams (95% C.I., 2.58-3.15); in 95 non-tumoral individuals, including 38 chronic non-malignant diseases plus 57 normal controls, 3.09 milligrams (2.46-3.72) and in malignant tumors, 4.12 milligrams (3.80-4.45). There was a statistically significant difference between chronic diseases and normal controls (P < 0.05) and also between cancer patients and non-tumoral individuals, normal control and chronic non-tumoral diseases (P < 0.001). The means of plasmatic A1AP by tumoral site are increasing in this order: breast, gastrointestinal, head and neck, and lung. The means by clinical stage are increasing in this order: complete remission, local disease, local-regional disease and metastatic disease. The calculated cutoff value, excluding complete remission cases, is 3.37 milligrams, with sensitivity 67.7% and specificity 67.7%. We conclude that there is an increase of plasmatic A1AP in cases of clinically active cancer compared with normal controls and normal range values in clinical complete remission. It can be an acceptable cancer marker that discriminates cancer from chronic non-tumoral diseases and complete clinical remission from relapses.

Utility of serum activity of angiotensin-converting enzyme as a tumor marker.

The objective of this study was to evaluate the diagnostic utility of the measurement of the serum activity of angiotensin-converting enzyme (SACE) as a cancer marker. This case-control study included 135 patients with cancer of different sites, confirmed histologically, and 145 controls (107 normal individuals plus 38 chronically ill patients with nontumoral diseases). Determination of SACE activity was done by a spectrophotometric method using as substrate the synthetic tripeptide N-(3-[2-furyl]acryloyl)-L-phenylalanylglycine. There were no sex- or age-related variations in SACE activity. Mean SACE activity in 107 normal controls was 51.6 U/l (95% C.I., 50.1-53.1); in 145 nontumoral individuals, including 38 chronic nonmalignant diseases plus 107 normal controls, 51.5 (50.1-53.1) and in malignant tumors 35.7 (32.8-38.5). There was no statistically significant difference between chronic diseases and normal controls (p > 0.05); but there was one between cancer patients and nontumoral individuals, normals and chronic nontumoral diseases. The mean of SACE activity values by tumoral site are (U/l; 95% C.I.): breast, 41.3 (36.2-46.5); gastrointestinal 31.5 (24.3-38.8); head and neck, 32.3 (26.7-37.8), and lung 27.6 (21.6-33.6) (p < 0.001). The means by clinical stage are: complete remission, 58.0 (53.7-62.3), significantly higher than in normal controls (p < 0.001); local disease, 40.56 (34.5-46.5); locoregional disease, 35.09 (30.7-39.4); metastatic disease, 23.04 (19.5-26.5), and in relapse at diverse stages, 30.86 (25.1-36.5). In clinical active cases, there is a statistically significant decrease of SACE activity, especially in metastatic disease (p < 0.001). The calculated cutoff value, excluding complete remission cases, is 40.7 U/l, with sensitivity of 69.5% and specificity of 91.6%. We conclude that there is a decrease of SACE activity in cases of clinically active cancer and an increase in clinical complete remission.