Evaluation of plasma membrane integrity of frozen-thawed goat spermatozoa with or without seminal plasma

The aim of the present work was to evaluate plasma membrane integrity, motility, vigor and morphology of fresh and frozen goat spermatozoa with or without seminal plasma. Semen samples were diluted in Tris solution, before and after thawing, with a combination of carboxifluorescein diacetate and propidium iodide. The results showed differences (P < 0.01) for motility and minor defects in the presence or absence of seminal plasma, for both fresh and frozen samples. Periods of collection had a significant effect on motility, probably due to changes in the photoperiod. Plasma membrane integrity was significantly reduced by the freezing process, whether seminal plasma was present or absent. In conclusion, removal of seminal plasma decreased motility and vigor rates in frozen samples. The photoperiod probably decreased the testosterone level, contributing negatively to the high percentage of sperm abnormalities, mainly damaged membranes. The use of fluorescent probes allowed a better estimation of the percentage of functional cells, instead of only estimating the percentage of motile cells or morphology defects. © 2001 Elsevier Science B.V. All rights reserved.

Spermiophagy in the spermatheca of Melipona bicolor lepeletier, 1836 (Hymenoptera, Apidae, Meliponini)

The presence of spermatozoa in vesicles in the cytoplasm of the epithelial cells that constitute the spermathecal wall of Melipona bicolor queen (Meliponini) is discussed in relation to the organ structure. The epithelial wall is lined by an apparently continuous cuticle in the luminal surface that should be a non-transposable barrier to the luminal spermatozoa. However, some spermatozoa were seen crossing the cuticle through interruptions that was first interpreted as sectioning defects. Nevertheless, the sperm cells in well-structured cytoplasmic vesicles, bound by membranes and sometimes associated to multivesicular bodies, as well as cytoplasmic structures representative of intracellular digestion and the occurrence of the phenomenon in two of the three spermathecae studied, suggest a real spermiophagic hole in the spermathecal epithelial cells.

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/ mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 μM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/ BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm. © 2003 American Society of Animal Science. All rights reserved.

Early healing pattern of autogenous bone grafts with and without e-PTFE membranes: A histomorphometric study in rats

Objective. The aim of this study was to perform quantitative and qualitative analyses of the initial repair pattern of an autogenous bone block graft when covered or not with e-PTFE membranes. Study design. Sixty male Wistar rats received a bone graft plus an e-PTFE membrane (MB) or just the graft (B). A block graft was harvested from the animal's calvarium and was laid and stabilized on the external cortical area near the angle of the mandible. Descriptive histology and histomorphometric analyses were carried out and the data were analyzed statistically by ANOVA and the Tukey test, with the level of significance set at 5%. Results. The results for group B showed that there was bone loss during the healing period (B0 = 1.38, B45 = 1.05, F = 7.91 > F C = 3.02), that is, the initial volume of the graft decreased in time. Bone tissue loss was about 24%. In contrast, the MB group showed bone tissue gain along the observation period (MB0 = 1.54, MB45 = 2.40, F = 7.91 > FC = 3.02), meaning that the total volume of newly formed bone was greater than the original graft area. Bone tissue gain was approximately 55%. MB showed significantly greater bone gain when compared to B (B45 = 1.05, MB45 = 2.40, F = 39.86 > FC = 1.90). These significant differences between B and MB could already be observed after 21 days. Conclusions. The bone block graft underwent resorption at an early healing stage, while additional new bone formation was observed when the bone graft was covered with an e-PTFE membrane. © 2005 Mosby, Inc. All rights reserved.

Decrease of the DNA damage levels after sperm preparation by layering method

The aim of this prospective study was to determine the DNA fragmentation levels before and after sperm preparation by layering method. A total of 78 patients submitted to assisted reproduction technology (ART) for infertility treatment were evaluated. Ejaculated spermatozoa were obtained by masturbation on the day of ART procedure. The evaluation of DNA fragmentation was performed in the fresh semen and after preparation by a layering method, respectively. After washing with PBS, the sperm pellets were smears and then processed for the terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) assay that was performed using a Cell Death Detection Kit with tetramethylrhodamine-labelled dUTP. For quantitative evaluation, 200 spermatozoa in randomly selected areas on microscope slides were evaluated and the percentage of TUNEL positive spermatozoa was determined. If ≥20% of selected sperm were TUNEL positive, the exam was considered abnormal. The mean percentage of DNA sperm fragmentation before sperm preparation was 17±8.3% and after 7.8±6.5% (p<0.0001). The exam was considered normal in 49 patients before preparation and in 73 patients after (p<0.0001). The sperm preparation with a layering method for the ART procedure is effective to select sperm with a significant decrease of the DNA damage.

The effects of male age on sperm DNA damage in an infertile population

The objective was to investigate the influence of age on sperm DNA damage. Semen samples were collected from 508 men in an unselected group of couples attending infertility investigation and treatment. DNA fragmentation in spermatozoa was measured by TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick-end labelling (TUNEL) assay; at least 200 spermatozoa in randomly selected areas of microscope slides were evaluated using a fluorescent microscope and the percentage of TUNEL positive spermatozoa was determined. The number of cells with red fluorescence (TUNEL positive) was expressed as a percentage of the total sample [DNA fragmentation index (DFI)]. Age was treated as a continuous variable for regression and correlation analysis. The following male age groups were used: Group I: ≤35 years, Group II: 36-39 years, and Group III: ≥40 years. DFI was significantly lower in Group I than in Group II (P = 0.034) or III (P = 0.022). There was no difference in DFI between Groups II and III. In addition, regression analysis demonstrated a significant increase in sperm DFI with age (P = 0.02). TUNEL assay clearly demonstrates an increase in sperm DNA damage with age. © 2007 Published by Reproductive Healthcare Ltd.

Reconstruction of Mandibular Segmental Defects Using the Guided-Bone Regeneration Technique With Polylactide Membranes and/or Autogenous Bone Graft: A Preliminary Study on the Influence of Membrane Permeability

Purpose: Bone maintenance after mandibular reconstruction with autogenous iliac crest may be disappointing due to extensive resorption in the long term. The potential of the guided-bone regeneration (GBR) technique to enhance the healing process in segmental defects lacks comprehensive scientific documentation. This study aimed to investigate the influence of polylactide membrane permeability on the fate of iliac bone graft (BG) used to treat mandibular segmental defects. Materials and Methods: Unilateral 10-mm-wide segmental defects were created through the mandibles of 34 mongrel dogs. All defects were mechanically stabilized, and the animals were divided into 6 treatment groups: control, BG alone, microporous membrane (poly L/DL-lactide 80/20%) (Mi); Mi plus BG; microporous laser-perforated (15 cm2 ratio) membrane (Mip), and Mip plus BG. Calcein fluorochrome was injected intravenously at 3 months, and animal euthanasia was carried out at 6 months postoperatively. Results: Histomorphometry showed that BG protected by Mip was consistently related to larger amounts of bone compared with other groups (P ≤ .0001). No difference was found between defects treated with Mip alone and BG alone. Mi alone rendered the least bone area and reduced the amount of grafted bone to control levels. Data from bone labeling indicated that the bone formation process was incipient in the BG group at 3 months postoperatively regardless of whether or not it was covered by membrane. In contrast, GBR with Mip tended to enhance bone formation activity at 3 months. Conclusions: The use of Mip alone could be a useful alternative to BG. The combination of Mip membrane and BG efficiently delivered increased bone amounts in segmental defects compared with other treatment modalities. © 2008 American Association of Oral and Maxillofacial Surgeons.

What's the signification of nuclear vacuoles in human sperm?

The aim of this study was to determine the extent of DNA fragmentation and the presence of single/denatured or double stranded of DNA in sperm with large nuclear vacuoles (LNV) selected by high-magnification. A total of 30 patients had fresh semen samples prepared by discontinuous concentration gradient. Sperm with normal nucleus (NN) and LNV were selected at 8400x magnification and placed in different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double stranded DNA was identified by acridine orange fluorescence method. The percentage of DNA fragmentation in LNV sperm (29%) was significantly higher (P<0.001) than NN sperm (15.8%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono and oligonucleosomes), and single strand breaks (nicks) in high molecular weight DNA occur more frequently in LNV. Identically, the percentage denatured stranded DNA in sperm with LNV (67.9%) was significantly higher (P <0.0001) than NN sperm (33%). The high level of denatured DNA in sperm with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibers. Our results support an association between LNV sperm and DNA damage, and the routine selection and injection of morphological motile sperm at high magnification for ICSI. The adverse effect (DNA fragmentation or denaturation) leads to concern particularly about the possibility of iatrogenic transmission of genetic abnormalities. Copyright - SBRA - Sociedade Brasileira de Reprodução Assistida.

Sperm motility of Prochilodus lineatus in relation to dilution rate and temperature of the activating medium

The objective of this study was to assess the effects of an activating solution on the sperm motility duration (SMD) of 'curimbatá', Prochilodus lineatus through of the definition of qualitative and quantitative parameters of the semen pool used in the experiment; evaluation of the effects of different ratios of semen dilution corresponding to 1-:-1, 1-:-2, 1-:-20, 1-:-200, 1-:-2000, 1-:-20-000 and 1-:-100-000 semen:dilute solution on the SMD and, assessment of the effects of different temperatures of the activating solution (5, 10, 15, 20, 25, 30, 35, 40, 45 and 50°C) on the SMD. The results of SMD were directly proportional to the dilution (P<0.05), starting from the dilution of 1-:-2 (semen:water), with 23.04-s. Were used three replicates of the semen pool for each test. Two-year-old brookstock were maintained in ponds culture conditions. In November-December 2006, twelve mature males broodfish were selected (mean weight and length of 405.8±134.2-g and 25.6±3.1-cm, respectively). The males released that semen under slight pressure of the urogenital papilla were selected for the experiment. The SMD increased proportionally to the increase in dilution, until it reached a maximum of 28.83-s for the ratio 1-:-100-000 semen: dilute solution. The results of SMD in relation to the temperature of the activating solution exhibited a quadratic behavior (P<0.05) with a maximum theoretical performance in terms of sperm motility duration of 21.36-s at a temperature of 17.3°C. Thus, for the species considered, the increase in the dilution ratio proved favorable for the rise in motility duration until the maximum value studied of 1-:-100-000 semen:dilute solution. As for the temperature of the activating solution, the best results of SMD were obtained at the temperature of 17.3°C. At higher temperatures used in the experiment (25, 30, 35, 40, 45 and 50°C), a decrease in motility duration. © 2010 Blackwell Verlag, Berlin.

Reproductive technology in domestic carnivorous

Background: The delay in development of artificial reproduction techniques on carnivorous could be due to countless reasons, but the lack of commercial interest is probably the most important one. The majority of canines are small structures, canidae are extremely fertile and a great number of species are adapted to domestication or captivity. Finally, the canine gamete physiology presents a difficult adaptation of technology knowledge obtained from other species. Furthermore, domestic felines are animals of company and there is no interest in reproducing them in a large scale, as it has been observed in other domestic animals, however, besides of being a valuable model for the development of in vitro techniques, the domestic cat is also used as an embryo receptor for different species of small wild felines due to physiological similarities among them, in vitro embrionary development, Review: It was reviewed the main insights about the reproductive physiology in female dogs, in vitro oocytary maturation (IVM), pregnancy and conception rate with dogs' frozen/unfrozen semen and PIV in domestic cats. The majority of mammal oocytes restart meiosis spontaneously after ovulation and reaches MII in artificial environment; in an in vitro maturation system in bovines, around 90% of oocytes complete their maturation, although its development capacity can be reduced subsequently. The success of IVM in canidae have been limited, with maturation rate varying from 0 to 58%, usually around 20%. The greatest difficulties include oocyte quality, hormonal environment, protein supplementation, cumulus / oocyte cell interaction, donor breed and age, culture systems, oxygen tension, amino acids, growth factor and sequential means. The freezing process reduces the quality of the semen, firstly because it reduces the number of living sperms and secondly because freezing produces cell modifications that could alter the sperm motility, longevity, integrity of membranes and its fertilizing capacity. Conclusion: Nowadays, several researches are being performed with the aim of increasing viability after dogs' and cats' semen is unfrozen, using extenders, cryoprotectors, freezing and unfreezing curves, addition of antioxidant substances. The aim of this text is to inform about the improvements obtained on the artificial reproduction techniques, emphasizing the oocytary maturation in female dogs, semen cryopreservation and artificial insemination in domestic dogs and cats.

Luminescent multifunctional biocellulose membranes

Luminescent biocellulose membranes were obtained by incorporation of ethanolic solutions of the europium compounds [Eu(BTFAC)3(H2O)2], [Eu(BTFA) 3(DBSO)2], [Eu(BTFA)3(PTSO)2] and [Eu(BTFA)3(FSO)2] (BTFAC- 4,4,4-Trifluoro-1- phenyl-1,3-butanedione DBSO- dibenzyl sulfoxide, PTSO- p-Tolyl sulfoxide and FSO- phenyl sulfoxide). Selfsustainable semi-transparent composite membranes were obtained showing strong emission under UV exctiation. The antenna hole played by the ligands was observed to be more efficient in the composite membranes than in the precursor complexes which by themselves are also strong red emitter compounds. These new multifuctional membranes could find application in different areas as phosphors and UV→Visible energy converting devices. © 2011 Copyright Society of Photo-Optical Instrumentation Engineers (SPIE).

Effect of Removing Seminal Plasma Using a Sperm Filter on the Viability of Refrigerated Stallion Semen

Cooling of equine semen obtained from some stallions results in lower seminal quality and viability when the seminal plasma (SP) is present. The objective of this study was to evaluate the effect of the removal of SP using a Sperm Filter on the viability of cooled stallion semen. For this purpose, 31 stallions were used. Their ejaculates were divided into three groups: CN, semen was diluted with an extender; FLT, SP was removed by filtration; and CT, SP was removed by centrifugation and cooled to 15°C for 24 hours. Sperm kinetics and plasma membrane integrity were evaluated immediately after collection (T0) and after 24 hours of refrigeration (T1). No difference (P > .05) was noted at T1 for total sperm motility (TM), progressive sperm motility, or plasma membrane integrity when semen samples from all the stallions were analyzed. However, when samples from stallions termed bad coolers were analyzed (TM = <30% at T1), a difference was observed in TM and progressive sperm motility for CN compared with FLT and CT at T1. Sperm recovery was greater when SP was removed using the filter (FLT) to that when the SP was removed by centrifugation (CN) (89% vs. 81%). Thus, we concluded that filtering with a Sperm Filter is an efficient and practical method for removal of SP from stallion ejaculates, with lower sperm loss than centrifugation. We also found that the presence of SP reduces the quality and viability of cooled semen from stallions whose semen is sensitive to the process of refrigeration. © 2013 Elsevier Inc.

When small is different: The case of membranes inside tubes

Recently, classical elasticity theory for thin sheets was used to demonstrate the existence of a universal structural behavior describing the confinement of sheets inside cylindrical tubes. However, this kind of formalism was derived to describe macroscopic systems. A natural question is whether this behavior still holds at nanoscale. In this work, we have investigated through molecular dynamics simulations the structural behavior of graphene and boron nitride single layers confined into nanotubes. Our results show that the class of universality observed at macroscale is no longer observed at nanoscale. The origin of this discrepancy is addressed in terms of the relative importance of forces and energies at macro and nano scales. © 2012 Materials Research Society.