Cryopreservation of epididymal sperm from ibexes (Capra pyrenaica) using short equilibration time with glycerol

Two experiments were conducted to study the effect of shortening the equilibration time with the cryoprotectant glycerol before freezing epididymal sperm recovered postmortem from Iberian ibex. In the first experiment, the standard equilibration time of 3 hours was compared with 2 hours, and subjective sperm motility and quality of movement were greater (P < 0.05) in the latter group. In the second experiment, reducing the equilibration time from 2 hours to 15 minutes did not affect sperm motility (evaluated subjectively and objectively), viability, acrosomal integrity, or membrane functional integrity. In conclusion, shortening the equilibration time can be used as a technique to simplify the cryopreservation process and this provides practical advantages under field conditions.     

Cryopreservation of Spanish ibex (Capra pyrenaica) sperm obtained by electroejaculation outside the rutting season

The effectiveness of electroejaculation for obtaining Spanish ibex sperm samples for freeze preserving outside the rutting season was evaluated—the aim being to optimise biological resources for the establishment of germplasm banks. The effect of different egg yolk concentrations (6% or 12%, v/v) in diluents of different buffer composition (Tris-citric acid buffer or Tes-Tris buffer) on frozen-thawed samples of the above also investigated. Experiments were undertaken with six ibex males in February-May, and involved four different semen samples from each animal with four combination of extender, respectively: Tes-Tris-glucose (TTG)-6% egg yolk, TTG-12% egg yolk, Tris-citric acid-glucose (TCG)-12% egg yolk, TCG-6% egg yolk. The results show that electroejaculation is a useful way of obtaining sperm samples from Spanish ibex outside the rutting season (i.e., at a time coinciding with plasma testosterone levels of <0.4 ng/ml). According to the results of the eosin-nigrosin staining and the hypo-osmotic swelling test, the freezing-thawing process significantly reduced the viability and membrane integrity of the spermatozoa extended with TTG-6% egg yolk, TTG-12% egg yolk, and TCG-12% egg yolk, but did not affect these variables in spermatozoa extended with TCG-6% egg yolk. Therefore, the use of Tris-citric acid-based extenders containing low concentrations of egg yolk is recommended for cryopreserving Spanish ibex spermatozoa obtained by electroejaculation outside the rutting season.     

Freezability of Iberian ibex (Capra pyrenaica) spermatozoa according to the glycerolization temperature and plasma testosterone concentration

Ibex spermatozoa can be successfully frozen using glycerolated media. However, no information is available regarding the most effective method of glycerol addition in this species. The aim of the present work was to evaluate the effect of the glycerolization temperature on the response to freezing-thawing of ibex spermatozoa collected by electroejaculation. The effect of the interaction glycerolization temperature x plasma testosterone concentration was also evaluated. The spermatozoa used in this work came from six adult ibexes maintained in captivity. Each ejaculate was divided into two aliquots in a Tris-egg yolk-based medium. One fraction was subjected to single step dilution with 5% glycerol at room temperature (23 °C). The other fraction was diluted in two steps, first by dilution at room temperature with an extender identical to that described above but without glycerol, followed by the addition of glycerol after cooling to 5 °C. The glycerolization temperature did not affect any sperm variable after thawing. Heterospecific artificial insemination involving domestic goats, revealed no differences in the fertilization rate for frozen-thawed spermatozoa diluted by the one or two step procedures (18.2% vs. 20.0%). The interaction glycerolization temperature x plasma testosterone concentration had no affect on the freezing-thawing of the sperm cells. The results revealed, however, that high plasma testosterone levels during the pre-rutting season may interfere with the freezing-thawing process, having a negative influence on sperm cryosurvival.     

Influence of recovery method and microbial contamination on the response to freezing–thawing in ibex (Capra pyrenaica) epididymal spermatozoa

The method of sperm recovery may influence the initial quality of sperm samples and their response to freezing–thawing. The aim of the present work was to compare two methods for collecting epididymal spermatozoa in order to improve the quality of recovered sperm and reduce possible contamination. Testes were obtained from 23 legally hunted, adult ibex males. The sperm mass of the right epididymis was collected by small longitudinal and transverse cuts made in the cauda epididymidis. The sperm mass of the left epididymis was collected by retrograde flushing from the vas deferens to the cauda epididymidis (using a cannula), employing a Tris, citric acid, glucose, egg yolk-based medium. The flushing method recovered more spermatozoa (P < 0.001) than the cutting method. After freezing–thawing, greater acrosomes damage (P < 0.001) and more morphological abnormalities (P < 0.05) were seen among the sperm cells recovered by the cutting method than among those obtained by retrograde flushing. The method of sperm recovery did not, however, influence the microbial contamination rate. In frozen–thawed samples that were microbially contaminated, motility was significantly reduced (P < 0.05) and membrane integrity tended to be poorer (P = 0.06). In conclusion, retrograde flushing is recommended for ibex sperm collection since it would appear that microbial contamination is no more of a problem than that encountered with the cutting method, while a larger number of sperm cells more resistant to freezing–thawing can be obtained.     

Sarcoptes scabiei: Specific immune response to sarcoptic mange in the Iberian ibex Capra pyrenaica depends on previous exposure and sex

Host acquired immunity is a critical factor that conditions the survival of parasites. Nevertheless, there is a shortage of data concerning inter-individual immunological inequalities in wild mammals. Sarcoptic mange is a widespread parasitosis that severely affects mammals such as the Iberian ibex (Capra pyrenaica). Despite some work on the subject, the immune response to sarcoptic mange infestation is still a complex and poorly understood phenomenon. To improve knowledge of the host-Sarcoptes immunological interaction, 18 Iberian ibexes were experimentally infested. IgG levels were assessed using ELISA to test for potential factors determining the specific immune response to infestation. Previous exposure and sex appeared to affect the IgG response to infestation and our results suggest a sex-biased immunomodulation. We discuss the immunological pattern of host-Sarcoptes interactions and also suggest further lines of work that may improve the understanding of immunological interactions of host-Sarcoptes systems.     

Teladorsagia circumcincta: Survival of adults in vitro is enhanced by the presence of a mammalian cell line

Adult Teladorsagia circumcincta survival and motility in vitro was examined in a range of different cell culture media, supplements and gas mixes. Under optimum conditions, worms survived for 14 days, exhibiting high motility for 9 days and egg production for 72 h. Optimum conditions involved co-culture of worms with a HeLa cell line in a supplemented cell medium (CEM) and an atmosphere containing 10% CO₂, 5% O₂ 85% N₂, 65% humidity at 37 °C. The incubation medium consisted of Minimum Essential Medium with 10% fetal calf serum, 1% non-essential amino acids, 1% glutamax and 1% penicillin-neomycin-streptomycin cocktail mix. Compared with optimum conditions, incubation in CEM alone, cell conditioned CEM, RPMI alone, Medium 199 alone, reduced CO₂ or O₂, or when cells were replaced with Escherichia coli, both survival and motility were reduced. Optimum conditions for adult T. circumcincta maintenance for culture, anthelmintic testing or generation of excretory/secretory products are described.     

Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization

Since the first successful reports into oocyte freezing, many papers concerning the cryopreservation of mouse oocytes have been published. However, a simple and practical cryopreservation method for unfertilized C57BL/6 mouse oocytes, and an IVF system using these cryopreserved oocytes have yet to be established, in spite of the fact that C57BL/6 is the prevalent inbred strain and is used for large-scale knockout programs. In this study, unfertilized C57BL/6 mouse oocytes were cryopreserved via a simple vitrification method. After warming, IVF was performed using cryopreserved unfertilized oocytes and fresh sperm, cryopreserved unfertilized oocytes and cold-stored sperm, cryopreserved unfertilized oocytes and frozen sperm (C57BL/6 strain sperm), and cryopreserved unfertilized oocytes and frozen sperm derived from GEM strains (C57BL/6 background GEM strains). Nearly all of the cryopreserved oocytes were recovered, of which over 90% were morphologically normal. Those oocytes were then used for in vitro fertilization, resulting in 72–97% of oocytes developing into 2-cell embryos. A portion of the 2-cell embryos were transferred to recipients, resulting in live young being produced from 32–49% of the embryos. In summary, we established the simple and practical method of mouse oocyte vitrification with high survivability and developmental ability and the IVF using the vitrified-warmed oocytes with fresh, cold-stored or cryopreserved sperm with high fertility.     

Influence of Aloe vera on water absorption and enzymatic in vitro degradation of alginate hydrogel films

This study investigates the influence of Aloe vera on water absorption and the in vitro degradation rate of Aloe vera-Ca-alginate hydrogel films, for wound healing and drug delivery applications. The influence of A. vera content (5%, 15% and 25%, v/v) on water absorption was evaluated by the incubation of the films into a 0.1 M HCl solution (pH 1.0), acetate buffer (pH 5.5) and simulated body fluid solution (pH 7.4) during 24 h. Results show that the water absorption is significantly higher for films containing high A. vera contents (15% and 25%), while no significant differences are observed between the alginate neat film and the film with 5% of A. vera. The in vitro enzymatic degradation tests indicate that an increase in the A. vera content significantly enhances the degradation rate of the films. Control films, incubated in a simulated body fluid solution without enzymes, are resistant to the hydrolytic degradation, exhibiting reduced weight loss and maintaining its structural integrity. Results also show that the water absorption and the in vitro degradation rate of the films can be tailored by changing the A. vera content.     

双色真藓的组织培养和发育研究

对双色真藓(Bryum dichotomum Hedw.)的孢子发育过程及愈伤组织的诱导和培养进行了研究。结果表明,双色真藓孢子萌发和原丝体发育属于典型的真藓型。将双色真藓原丝体接种在含有2.0 mg L-1的硅酸钠和3.0 mg L-1 6-BA的MS固体培养基上,可诱导双色真藓原丝体分化为愈伤组织。愈伤组织在含有2.0 mg L-1的硅酸钠、1.0 mg L-12,4-D和1.0 mg L-1 6-BA的MS固体培养基上可以长期继代培养。而愈伤组织在含有2.0 mg L-1的硅酸钠、1.0 mg L-1 2,4-D和1.0 mg L-1 6-BA的MS液体培养基中可以悬浮培养,且生长迅速,培养28 d达到接种鲜重的9.25倍。     

Reproducible reference frame for in vitro testing of the human vertebrae

Definition of an anatomical reference frame is necessary for in vitro biomechanical testing. Nevertheless, there is neither a clear recommendation, nor consensus in the literature concerning an anatomical reference frame for in vitro testing of the human vertebrae. The scope of this work is to define a reference frame for the human vertebrae for in vitro applications. The proposed anatomical reference frame relies on alignment of well-defined points on the endplates, and on two landmarks on the posterior wall. The repeatability of the proposed alignment procedure has been tested in vitro by 5 operators, on 7 specimens. Furthermore, the feasibility and repeatability of the proposed procedure was assessed in silico, using CT-scans of the same specimens.     

Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization,perivitelline, and intracytoplasmic sperm injections

IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine sperm capacitated with dilauroyl phosphatidylcholine would result in higher fertilization rates than standard IVF in bovine and equine oocytes. In experiment 1, bovine oocytes were used for (1) IVF with bovine sperm, (2) IVF with equine sperm, and (3) intracytoplasmic sperm injections (ICSIs) with equine sperm. Presumptive zygotes were either stained with 4′,6-diamidino-2-phenylindole from 18 to 26 hours at 2-hour intervals or evaluated for cleavage at 56 hours after addition of sperm. Equine sperm fertilized bovine oocytes; however, pronuclei formation was delayed compared with bovine sperm after IVF. The delayed pronuclear formation was not seen after ICSI. In experiment 2, bovine oocytes were assigned to the following five groups: (1) cumulus oocyte complexes (COCs) coincubated with bovine sperm; (2) COC exposed to sucrose then coincubated with bovine sperm; (3) COC coincubated with equine sperm; (4) COC exposed to sucrose, and coincubated with equine sperm; and (5) oocytes exposed to sucrose, and 10 to 15 equine sperm injected into the perivitelline (PV) space. Equine sperm tended (P = 0.08) to fertilize more bovine oocytes when injected into the PV space than after IVF. In experiment 3, oocytes were assigned to the following four groups: (1) IVF, equine, and bovine COC coincubated with equine sperm; (2) PVI of equine and bovine oocytes; (3) PVI with equine oocytes pretreated with sucrose; and (4) ICSI of equine oocytes. Oocytes were examined at 24 hours for cleavage. No equine oocytes cleaved after IVF or PVI. However, ICSI conducted with equine sperm treated with dilauroyl phosphatidylcholine resulted in 85% of the oocytes cleaving. Sperm injected into the PV space of equine oocytes did not appear to enter the ooplasm. This study validated the use of bovine oocytes for equine sperm studies and indicates that failure of equine IVF is more than an inability of equine sperm to penetrate the ZP.     

Entamoeba histolytica, heterologous expression and in situ immunolocalization of ornithine decarboxylase (EhODC)

Previous studies from this laboratory have dealt with the purification and biochemical characterization of ornithine decarboxylase (ODC) from Entamoeba histolytica. Enzyme compartmentalization has been described as a major mechanism in the regulation of polyamine metabolism. However, the subcellular location of ODC in the human parasite has remained unresolved. To examine this issue, we cloned the full-length gene (Ehodc) encoding for the parasite enzyme, whose open reading frame encodes for a peptide of 412 amino acids with an estimated molecular mass of 46 kDa that exhibits similarity to other ODCs. Heterologous overexpression of the gene allowed us to purify the recombinant protein (rEhODC) by metal affinity chromatography. The purified polypeptide was used to raise heteroclonal antibodies that were utilized to localize the enzyme in situ by immunofluorescence and confocal microscopy. EhODC was observed to be associated with the plasma membrane, in vesicles close to the plasma membrane and in the EhkOs organelle.     

Reproducible and controllable light induction of in vitro fruiting of the white-rot basidiomycete Pleurotus ostreatus

Fruiting is a crucial developmental process in basidiomycetes yet the genetic and molecular factors that control it are not yet fully understood. The search for fruiting inducers is of major relevance for both basic research and for their use in industrial applications. In this paper, an efficient and reproducible protocol for controlled fruiting induction of Pleurotus ostreatus growing on synthetic medium is described. The protocol is based on the control of light intensity and photoperiod and permits the life cycle for this fungus to be completed in less than two weeks. The fruiting bodies produced by this method release fertile spores after 4-5 d of culture. Our results indicate that fruiting induction is solely dependent on the illumination regime and that it occurs long before the available nutrients are depleted in the culture. This protocol will greatly facilitate molecular and developmental biology research in this fungus as it avoids the need for complex culture media based on lignocellulosic materials or the use of chemical inducers.     

The influence of washing Spanish ibex (Capra pyrenaica) sperm on the effects of cryopreservation in dependency of the photoperiod

Extenders containing low concentrations of egg yolk are recommended for cryopreserving ibex spermatozoa. However, the phylogenetic relationship of the Spanish ibex (Capra pyrenaica) with domestic goats suggests that phospholipases in the seminal plasma may have a negative effect on the response to freezing-thawing when egg yolk-based diluents are employed. The aim of the current work was to determine how seminal plasma removal from Spanish ibex semen, collected by electroejaculation over a period of 1 yr, affects its response to freezing-thawing. Semen was collected from six adult ibexes maintained in captivity. The negative effects of freezing-thawing on the quality of sperm motility and on the integrity of the acrosome and plasma membrane were more serious in the nonwashed semen samples than in those from which the seminal plasma had been removed (P < 0.01, P < 0.05, and P < 0.05 respectively). The beneficial effect of removing the seminal plasma was particularly noticeable during the time of decreasing photoperiod. This suggests that ibex semen shows increased phospholipase activity during the rutting season.     

Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

Synthesis, characterization and in vitro cytotoxicity studies of platinum(IV) complexes with thiouracil ligands

Reactions of [PtMe₃(bpy)(Me₂CO)][BF₄] (2) with the thionucleobases 2-thiouracil (s²Ura), 4-thiouracil (s⁴Ura) and 2,4-dithiouracil (s²s⁴Ura) resulted in the formation of complexes of the type [PtMe₃(bpy)(L-κS)][BF₄] (L = s²Ura, 3; s⁴Ura, 4; s²s⁴Ura, 5). The complexes were characterized by NMR spectroscopy (¹H, ¹³C, ¹⁹⁵Pt), IR spectroscopy as well as microanalyses. The coordination through the C4S groups (4, 5) was additionally confirmed by DFT calculations, where it was shown that these complexes [PtMe₃(bpy)(L-κS⁴)]⁺ (L = s⁴Ura, s²s⁴Ura) are about 5.8 (4b) and 3.3 kcal/mol (5b), respectively, more stable than the respective complexes, having thiouracil ligands bound through the C2X groups (X = O, 4a; S, 5a). For [PtMe₃(bpy)(s²Ura-κS²)][BF₄] (3) no preferred coordination mode could be assigned solely based on DFT calculations. Analysis of NMR spectra showed the κS² coordination. In vitro cytotoxic studies of complexes 3−5 on nine different cell lines (8505C, A253, FaDu, A431, A549, A2780, DLD-1, HCT-8, HT-29) revealed in most cases moderate activities. However, 3 and 5 showed significant activity towards A549 and A2780, respectively, possessing IC₅₀ values comparable to those of cisplatin. Cell cycle perturbations and trypan blue exclusion test on cancer cell line A431 using [PtMe₃(bpy)(s²s⁴Ura-κS⁴)][BF₄] (5) showed induction of apoptotic cell death. Furthermore, the reaction of [PtMe₃(OAc-κ²O,O′)(Me₂CO)] (6) with 4-thiouracil yielded the dinuclear complex [(PtMe₃)₂(μ-s⁴Ura_-H)₂] (7), which has been characterized by microanalysis, NMR (¹H, ¹³C, ¹⁹⁵Pt) and IR spectroscopy as well as ESI mass spectrometry. X-ray diffraction analysis of crystals yielded in an isolated case exhibited the presence of a hexanuclear thiouracilato platinum(IV) complex, possessing each three different kinds of methyl platinum(IV) moieties and 4-thiouracilato ligands. This exhibited the ability of 4-thiouracil platinum(IV) complexes to form multinuclear complexes.     

Synthesis, characterization and in vitro cytotoxicity of the first palladium(II) oxalato complexes involving adenine-based ligands

The first [Pd(Lⁿ)₂(ox)] xH₂O oxalato(ox) complexes involving 2-chloro-N6-(benzyl)-9-isopropyladenine (L¹; complex 1), 2-chloro-N6-(4-methoxybenzyl)-9-isopropyladenine (L²; 2), 2-chloro-N6-(2,3-dimethoxybenzyl)-9-isopropyladenine (L³; 3), 2-chloro-N6-(2,4-dimethoxybenzyl)-9-isopropyladenine (L⁴; 4), and 2-chloro-N6-(4-methylbenzyl)-9-isopropyladenine (L⁵; 5) have been synthesized by the reactions of potassium bis(oxalato)palladate(II) dihydrate, [K₂Pd(ox)₂]·2H₂O, with the mentioned organic compounds (H₂ox = oxalic acid; x = 0 for 1-3 and 5 or 2 for 4). Elemental analyses (C, H, N), FTIR, Raman and NMR (¹H, ¹³C, ¹⁵N) spectroscopies, conductivity measurements and thermal studies (thermogravimetric and differential thermal analyses, TG/DTA) have been used to characterize the prepared complexes. The molecular structures of [Pd(L²)₂(ox)] (2) and [Pd(L⁵)₂(ox)]·L⁵·Me₂CO (5·L⁵·Me₂CO) have been determined by a single crystal X-ray analysis. The geometry of these complexes is slightly distorted square-planar with two appropriate Lⁿ (n = 2 or 5) molecules mutually arranged in the head-to-head (2) or head-to-tail (5) orientation. The Lⁿ ligands are coordinated to the central Pd(II) ion via the N7 atoms. The same conclusions regarding the binding properties of L¹-L⁵ ligands can be made based on multinuclear NMR spectra. In vitro cytotoxicity of the complexes 1-5 has been evaluated against human chronic myelogenous leukaemia (K562) and human breast adenocarcinoma (MCF7) cancer cell lines. Significant cytotoxicity has been determined for the complexes 3 (IC₅₀ = 6.2 μM) and 5 (IC₅₀ = 6.8 μM) on the MCF7 cell line, which is even better than that found for the well-known and widely-used platinum-bearing antineoplastic drugs, i.e. oxaliplatin and cisplatin.     

Production of female bovine embryos with sex-sorted sperm using intracytoplasmic sperm injection: Efficiency and in vitro developmental competence

The production of embryos with a preselected sex sperm is important in the livestock industry. In this study, we examined the efficiency of producing female embryos by intracytoplasmic sperm injection (ICSI) with flow cytometry sorted (ssICSI) and unsorted (usICSI) bovine sperm, and their developmental competence in vitro. For comparison, bovine embryos were also produced by in vitro fertilization (IVF) with sorted (ssIVF) and unsorted (usIVF) bovine sperm. The semen used in this study was from a bull selected for its high fertility and blastocyst developmental competence among four bulls. We first examined and compared pronuclear (PN) formation and cleavage rates of the produced embryos among the treatment groups. Our results demonstrated that PN formation rates (judged by two pronucleus [2PN]) and cleavage rates in ssIVF group (23.1% and 43.6%) were lower than those in the usIVF (71.1% and 71.6%), usICSI (73.1% and 92.8%) and ssICSI (75% and 79.1%) groups, respectively (P < 0.05). Moreover, the blastocyst formation rate in the ssIVF group was less than those in the usIVF, usICSI, and ssICSI groups (2.7% vs. 30.2%, 28.7% and 24.7%, respectively; P < 0.05). Importantly, we reported that the blastocyst formation rate in the ssICSI group was similar to that in the usICSI group, which indicated that ICSI can rescue the damage introduced to sperm by flow cytometry–mediated sex-sorting. Of note, we achieved a blastocyst formation rate in the ssICSI group to be comparable with the usIVF group. We then examined embryo quality by counting the number of normal and apoptotic cells in blastocysts. It was found that, despite the fact that blastocyst formation rate in the ssIVF group was significantly lower than those in the usIVF, usICSI and ssICSI groups, there was no difference in total and apoptotic cell numbers among these groups (P > 0.05). Finally, karyotyping analysis demonstrated that the proportion of female embryos in the ssICSI and ssIVF groups was 100%, whereas it was 58.8% and 57.8% in the usIVF and usICSI groups, respectively. In conclusion, ICSI with flow cytometry sorted bovine sperm provides an alternative approach to produce embryos with predetermined sex.