Alternative procedures for the cryopreservation of brown bear ejaculates depending on the flexibility of the "in cooling" period (5°C).

The adaptability of cryopreservation protocols for brown bear spermatozoa collected under field conditions and frozen in a nearby laboratory (transported for a few hours) or shipped to a reference laboratory for sex sorting (transported for a few days) was evaluated. Forty-nine electroejaculates from 15 mature brown bears were extended to 100×10(6) sperm/mL in a TES-Tris-Fructose based extender and cryopreserved (-20°C/min to -100°C and stored at -196°C). After thawing, the quality of the seminal samples was assessed for total (TM), progressive (PM) motility and kinetic parameters - by CASA -, and viability (VIAB), viable and non-apoptotic status (YOPRO-), high membrane mitochondrial potential (MIT) and intact acrosomes (iACR) - by flow cytometry -. In Experiment 1, we assessed different storage times (0, 0.5, 1 - control -, 4-5, 7-8 and 11-12 h) at 5°C from final dilution to freezing. After thawing, non-equilibrated samples (0 h) showed lower values of iACR, TM and PM. No significant differences were found for the different periods of equilibration tested. In Experiment 2, we evaluated three long-term storage times (24, 48 and 72 h) at 5°C before freezing using storage for 1h as control. The post-thawing quality of brown bear spermatozoa declined markedly after 48-72 h of pre-freezing. In conclusion, our findings suggest the possibility of extending the pre-freezing cooling period up to 24h post-collection without freezing. This knowledge should enable the adaptation of the freezing protocols for when a special handling conditions are required such as the shipment of seminal samples to technological centers for the pre-freezing application of enhancer spermatic biotechnologies.

Effect of length of time post-mortem on quality and freezing capacity of Cantabric chamois (Rupicapra pyrenaica parva) epididymal spermatozoa.

Genome Resource Banks are keystones in the ex-situ conservation of wild species. Post-mortem (PM) collection of epididymal spermatozoa is an opportunistic and valuable source of germplasm, the time from the death of the animal limits its use. Seeking to improve germplasm preservation strategies for the chamois (Rupicapra sp.), the effect of PM time on epididymal sperm quality and freezability was studied using the Cantabrian chamois. Samples were classified according to PM collection time, up to 216 h (refrigerated), and cryopreserved (Tris-citric acid-fructose, 430 mOsm/kg, 15% egg yolk, 8% glycerol; freezing at -20 °C/min). Sperm quality was assessed after recovery and post-thawing (motility by CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). The sperm mass pH and osmolality showed a positive correlation with time. Total sperm motility dropped after 2 days PM, with progressivity and sperm velocities remained similar up to 3 days PM. Sperm freezability was acceptable, with the post-thawing HOST, motility, progressivity, VAP, VCL, VSL and BCF negatively correlating with PM time. Overall, chamois epidydimal samples were not adequate for preservation after 6 days PM. Freezability capacity could make these spermatozoa suitable for specific ART even if kept refrigerated for several days PM.

Influence of breed and age on morphometry and depth of inseminating catheter penetration in the ewe cervix: a postmortem study.

A detailed examination of the cervical canal in the ewe was carried out. This analysis could be used to design new catheters for artificial insemination (AI) to achieve deeper cervical penetration and therefore better fertility results. Three hundred and sixty-five cervices from four sheep breeds (Churra, Assaf, Merino, Castellana) obtained postmortem were used. Cervix morphometry and depth of cervical penetration using two types of catheters were determined. A conventional straight catheter for ovine artificial insemination (IMV), and a bent catheter, ending in a stainless steel needle, 9 cm in length and with an 8 mm tip bent 45 degrees , were used. The results showed that the morphometry of the cervix depends on breed and age of the ewe. The cervices of Churra breed were shorter and narrower, and had a higher number of folds than those of other breeds. Postmortem cervical penetration was deeper when the cervices were longer and wider, and with fewer folds (Merino and Castellana breeds). In ageing ewes, the cervix tended to become longer and wider, with loose folds. This decreased structural complexity and significantly improved cervical penetration. The bent catheter allowed significantly greater cervical penetration than the straight IMV one.

Organelle distribution in chick neuroepithelial cells: effects of colchicine and cytochalasin B.

The intracellular distribution of mitochondria, cytoplasmic inclusions and rough endoplasmic reticulum cisternae of chick neuroepithelial cells was investigated at neurulation stages 6, 8, 10 and 12. These neuroepithelial cells were subdivided into three zones: apical, median and basal and the distribution percentages of distribution of these organelles were obtained. Mitochondrial distribution was related to the energy supply that mitochondria provide for apical microfilament contraction. Cytoplasmic inclusions were distributed preferentially in the apical zone of the neuroepithelial cells during the four stages. Rough endoplasmic reticulum cisternae were homogeneously distributed in the three zones at stages 10 and 12, but at stages 6 and 8 there are more elevated percentages of rough endoplasmic reticulum in the apical zones than in the other zones. Experimental treatments with colchicine and cytochalasin B does not modify the patterns of mitochondria and rough endoplasmic reticulum cisternae but alters the distribution of cytoplasmic inclusions. Finally, there is a correlation in the normal neurulating neuroepithelial cells between the distributions of mitochondria and rough endoplasmic reticulum distribution and between the distributions of mitochondria and cytoplasmic inclusions distribution. This relationship is retained in the treated neuroepithelial cells.

Scanning electron microscopy of the aggregation of head mesoderm cells from chick embryos.

Head mesoderm cells from chick embryos at different stages of development were dissociated and cultured on plastic coverslips. In all cultures several cellular aggregates were described by means of scanning electron microscopy. Isolated cells present filopodia and lamellipodia. However, when mesoderm cells make contact with one another the filopodial and lamellipodial activity in the contact cellular edge disappear. Thus, the cells into cellular clusters do not present projections. The clusters were circular and bidimensional in character. The scanning electron microscopic observations showed that it is the type 1 variant of "contact inhibition of locomotion" which occurs. By means of these mechanisms the bidimensional aggregates are formed and cellular overlapping is not present. Since the behaviour of the mesoderm cells "in vitro" in some way could be comparable to their behaviour "in situ", the results here observed are discussed in relation to the conduct of mesoderm cells "in vivo".

Scanning electron microscopy of the wild boar and pig lingual papillae.

Lingual papillae of wild boar and pig were studied by means of scanning electron microscopy (SEM). Vallate papillae appear with the typical circumvallate morphology. Their papillary bodies show conical or fungiform-like and spicule-like pseudopapillae in both animals. Taste pores were seen in the papillary grooves. Microplicae or pits are visible at high magnification. In pig and wild boar similar foliate papillae were observed. Pig has less but wider leaves than wild boar. Taste pores on papillary walls were viewed. At high magnification microplicae were seen. Morphologically, fungiform papillae correspond with their denomination. Taste pores open onto the upper surface and they are easily identifiable by SEM. The rostral and lateral regions contain the major number of fungiform papillae. The lateral papillae of wild boar and pig show a high number of pores per papilla. These regions must be considered important in taste sensitivity. Lateral papillae in both animals could provide a source of taste buds for study. In both animals the fungiform papillary epithelium showed a pitted appearance as a consequence of keratinization by food environmental stress. The filiform papillae can be both simple and compound (with body and hairs). Large conical papillae are located caudally and curved in the same direction. Filiform and conical papillae have a function in food mastication, handling and deglutition.

Quantitative ultrastructural changes of the endodermal cells in the early chick embryo analysed by stereological methods.

The ultrastructure of endoderm cells of the area pellucida has been analysed in the chick embryo by stereological methods. These cells show a specific subcellular evolution which can be correlated with several aspects of morphogenetic behaviour. The cell form coefficient (CFc) changes notably from stage 5 (0.683) to stage 8 (0.446) accompanying the transformation of this layer into a squamous epithelium. An increase of the nuclear surface density is observed and is discussed in relation to the control of nucleocytoplasmic interchange. The mitochondrial volume and surface densities remain constant (3.12% of cellular volume and 0.727 mitochondria/mu(3) respectively). The endodermal cells possess higher levels of vitelline reserves (lipid bodies, 6.97% and yolk droplets, 8.90%) than other cellular types of the chick embryo. This fact is discussed with respect to the role of the endoderm in the phagocytosis of yolk. The RER length density shows an increase that could be related to some specific changes of the extracellular matrix during this period, but this fact remains to be demonstrated in relation to changes of Golgi membranes.

Field and in vitro assay of three methods for freezing ram semen.

Glycerol has been the most widely used cryopreservation agent for spermatozoa and a wide range of factors affect its action on sperm viability and fertilizing capacity. We tested three methods for freezing ram semen packed in 0.25 ml straws (final cellular concentration: 100 x 10(6) spz/ml). Method M1: Two-thirds of the final volume of diluent was added as solution A (without glycerol) to the pure semen at 35 degrees C. The sample was cooled to 5 degrees C (-0.30 degrees C/min), one-third of final diluent volume was added as solution B (final concentration of glycerol 4%) and the sample was maintained at 5 degrees C for 2h. It was then frozen in a programmable biofreezer (-20 degrees C/min down to -100 degrees C). Method M2: The sample was diluted with a specific solution at 35 degrees C (final concentration of glycerol 3%), cooled to 5 degrees C (-0.20 degrees C/min) and left for 2h. After that, it was frozen in nitrogen vapours. Method M3: Semen was diluted 1:1 in a specific solution (concentration of glycerol 2%) and cooled to 5 degrees C (-0.25 degrees C/min). The sample was then diluted again in the same solution to the final cellular concentration (final concentration of glycerol 4%). It was left for 1h at 5 degrees C and then frozen in a programmable biofreezer (-20 degrees C/min down to -100 degrees C). Best total motility (TM) and progressive motility (PM) (75.8 and 55.18%) were obtained using Method M3. Methods M1 and M3 gave significantly higher values (P<0.05) for kinetic parameters: average path velocity (VAP) (81.3 and 85.2 microm/s), straight-line velocity (VSL) (72.8 and 77.3 microm/s) and linearity (LIN) (66.6 and 68.8%). Method M2 showed the lowest kinetic parameters of motility (VAP 74.4, VSL 67.3 and LIN 62.5) and the highest percentage of cells with damaged plasma membrane (53.8%). Method M1 gave the worst results in viability and acrosome status assessed using fluorescence probes (31.3%-dead cells with damaged acrosomes-versus 25.4% in M2 and 23.3% in M3). A field trial carried out on fertility showed a significantly higher percentage of pregnant or lambing ewes (P<0.05) with Method M3 (67.3% versus 51.1% for M1 and 58.8% for M2). We concluded that the use of a simple dilution medium (test-fructose-glycerol-egg yolk) with the addition of glycerol (to 2% at 35 degrees C and to 4% at 5 degrees C) in two steps together with a programmable biofreezer was a productive method for freezing ram semen.

Sheep embryo cryopreservation by vitrification and conventional freezing.

The survival of ovine embryos (morulae and blastocysts) either frozen by a conventional method or vitrified was investigated in culture. In Experiment I, embryos were vitrified using a solution containing 25% propylene glycol and 25% glycerol. A group of embryos (simulated control) was processed without freezing to evaluate the toxicity of the vitrification solution. In Experiment II, embryos were exposed to a solution of PBS containing 10% glycerol and 0.25 M sucrose placed horizontally in a programmable freezer. Automatic seeding was applied at -7 degrees C in 2 positions on straws and cooled at -0.3 degrees C/min to -25 degrees C and then stored in liquid nitrogen. In vitro development rates of vitrified embryos were 12% (morulae) and 19% (blastocysts). Simulated embryos showed a higher rate of survival than embryos cryopreserved by vitrification (67 and 63%, morulae and blastocysts respectively). In conventional cooling, the blastocysts showed the highest viability percentage (67%) of all the experimental groups but these values decreased significantly in morulae (31%). Differences in temperature between straws placed in distinct positions in the freezing chamber and thermic deviation were observed when automatic seeding was applied. Embryo viability differed from 51 to 75% according the relative position of the embryos within the chamber. Survival was higher when automatic seeding was applied on the meniscus of the embryo column versus the central point of this column (65 vs 21%). The damage of both cryopreservation methods on zona pellucida integrity (27 and 35% in vitrified and conventionally frozen embryos, respectively) had no effect on the in vitro survival.

Ultrastructural and cytochemical comparison between calf and cow oocytes.

The use of prepubertal females (calves) to obtain oocytes for in vitro fertilization (IVF) programs, is being analyzed currently. This will increase the availability of female oocytes and will allow a reduction of the interval between generations. Differentials in the development capability of calf and cow oocytes have been assessed by different authors, establishing several ultrastructural and metabolic differences between them. This paper analyzes the morphometric and cytochemical differences between calf and cow oocytes through microscopic techniques. The oocytes morphologically classified as good are processed for electron microscopy a) in Epon 812 epoxy resin for morphometric analysis or b) in low temperature Lowycril K4M resin for cytochemical evaluation using Con A, GS, LPA, UEA, and WGA lectins marked with colloidal gold as probes. Calf oocytes show a greater density of microvilli on their surface and a greater number of endocytosic vesicles than those of the cow. On the other hand, cow oocytes show a larger superior mitochondrial population. In the cumulus cells it can be seen that calf oocytes have a greater volume of lipid droplets. Cytochemical analysis shows that calf oocytes have lectin marking restricted to the plasmic membrane, highlighting the presence of LPA. In cow oocytes, lectin marking can be seen both on the plasmic membrane and in the vacuoles, in both cases, with the LPA highlighted. In the zona pellucida of calf and cow oocytes, the same sugars appear (GS, LPA, WGA), and marking with LPA is more extensive in cow oocytes.

Salvaging urospermic ejaculates from brown bear (Ursus arctos).

The objective of this study was to reverse the osmotic stress of sperm in urine contaminated bear ejaculates that were obtained by electroejaculation using pre-freezing washing or density gradient centrifugation isolation. In Experiment 1, ejaculates were divided into six aliquots, five were diluted in each washing extender: 200, 300, 400, 500 and 700 mOsm/kg (prepared from a Tes-Tris-Fructose base, adding water or fructose as corresponds), at a 1:2 ratio (raw semen: washing solution, v/v); and the other aliquot was handled without washing (Control group). Samples were centrifuged at 600 × g for 6 min prior to freezing. In Experiment 2, ejaculates were divided into two aliquots: one was diluted 1:1 with TCG (Tris-Citric acid-Glucose) and centrifuged at 600 × g for 6 min (Centrifugation Control; C-Control); the other was treated with PureSperm density gradient column. After treatments, samples were cryopreserved. Sperm motility, viability (SYBR-14/propidium iodide (PI)) and acrosomal status (peanut agglutinin-fluorescein isothiocyanate (PNA-FITC)/PI) were analyzed before and after freezing. Ejaculates with an initial osmolality of less than 120 mOsm/kg treated with pre-freezing washing, and the Control sample had greater pre-freezing sperm motility than the raw ejaculate, but sperm viability was not different among these groups. The samples washed with 700 mOsm/kg solutions had the least pre-freezing viability. In the post-thawing evaluation, pre-freezing washing treatments did not provide any improvement in comparison with the Control sample, and treatment with 700 mOsm/kg extender had deleterious effects in all urospermic samples. PureSperm density gradient centrifugation applied to urospermic raw semen was suitable for improving sperm motility and viability of pre-freezing samples and the selected spermatozoa had greater freezing capacity.

Evaluation of ram semen quality using polyacrylamide gel instead of cervical mucus in the sperm penetration test.

Fertility is a very complex biological function that depends on several properties of the spermatozoa, including sperm motility. Two objectives are analyzed in this study: (1) Replace the cervical mucus by a synthetic medium in a sperm penetration test, and (2) evaluating the results of this test objectively analyzing the sperm number that migrates. In experiment 1, we have tested eight concentrations of acrylamide (1%-2%). Rheological properties of media were analyzed. The plastic straws, loaded with acrylamide, were placed vertically on the semen sample tube for 15 min at 39 °C. After, the acrylamides were placed, by segments of 5 mm, into wells of a 24-well plate, dyed with Hoechst 33342 and the number of spermatozoa were calculated by automated microscopy analysis. The 1.55% and 1.6% acrylamide gel showed a number of spermatozoa emigrating closer to that seen with natural mucus. In experiment 2, we applied the sperm penetration in acrylamide 1.6% and 1.55% using fresh semen and cooled semen at 15 °C and 5 °C. The spermatozoa counts were performed for each segment of 10 mm. Semen chilled at 15 °C presented intermediate values of sperm counts in comparison with fresh semen (higher) and 5 °C chilled semen. The sperm counts do not differ between acrylamides but the rheological properties of acrylamide 1.6% were more similar to those of the natural cervical mucus. In experiment 3, we have observed significant correlations between the number of spermatozoa and several sperm quality parameters (positive: progressive motility and velocity according to the straight path; negative: damaged acrosomes and apoptotic cells) in 1.6% acrylamide media. We conclude that the size of the cell subpopulation, objectively calculated, that migrate beyond 20 mm in 0.5-mL straws filled with acrylamide is a useful parameter in ram sperm quality assessment and further studies are needed to evaluate its relationship with field fertility.

Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine, and PureSperm 100) in preparation of fresh or frozen-thawed brown bear spermatozoa.

Centrifugation is a crucial procedure in sperm cryopreservation protocols of brown bear (Ursus arctos), because the semen must be processed to increase sperm concentration and/or clean urine-contaminated samples. The efficacy of three media for centrifugation (Maxifreeze [IMV technologies, L'Aigle, France], Cushion Fluid Equine (Minitübe, Tiefenbach, Germany), and PureSperm [Nidacon, Gothenburg, Sweden]) on the quality of bear spermatozoa was evaluated. In experiment one, two cushioned media used for protecting against mechanical stress during centrifugation were analyzed. In experiment two, a density gradient based on PureSperm was assessed in relation to the maximum retrieval and the quality of fresh spermatozoa, and the freezability of the spermatozoa selected in this density gradient was studied in experiment three. Finally, the selection of frozen-thawed sperm using PureSperm was analyzed in experiment four. Our results indicate that the use of dense isotonic cushion solutions (Maxifreeze, Cushion Fluid Equine) in centrifugation did not improve the quality of recovered spermatozoa compared with standard centrifugation. However, a density gradient prepared with PureSperm improved the quality of spermatozoa in fresh semen and frozen-thawed semen, but the spermatozoa selected from the fresh sample with this density gradient did not show a better resistance to freezing with this density gradient in comparison with the control sample.

Design and "in vivo" evaluation of two adapted catheters for intrauterine transcervical insemination in sheep.

In order to obtain better fertility, we evaluated two ovine artificial insemination (AI) catheters that were manufactured according to the anatomical structure of the ewe cervix. Morphometric data of the cervix in Churra and Assaf breeds were used to design two types of curved catheters: CAT06 with one curvature and ZIGZAG with five curvatures in a zigzag shape. Two commercial catheters (IMV(®) and Minitüb(®)) were used as controls. In experiment 1, cervical penetration and the degree of reflux were measured in a Cervical AI simulated assay both Churra (n=28) and Assaf ewes (n=28). In experiment 2, a fertility study was performed with three catheters (only one commercial control catheter - IMV) in 465 inseminations (Assaf); and a second study analyzed only the top two catheters (IMV and CAT06) in 428 inseminations (210 Assaf and 218 Churra). The ewes were synchronized using intravaginal sponges (40 FGA mg during 14 days) and 500 IU of eCG. Deeper penetration of the cervix was obtained with the new catheters compared with the commercial ones (1.5, 1.3, 3.5 and 3.2 cm for the IMV, Minitüb, CAT06 and ZIGZAG catheters, respectively). The cervical penetration and the reflux grade of each catheter showed no differences between breeds. In experiment 2, the degree of penetration had no correlation with fertility of different catheters. The best percentage of lambing ewes was obtained with the IMV and CAT06 catheters (39.5 and 48.1%, respectively. vs 27.2% for ZIGZAG catheter, in the Assaf breed). Regarding effect of breed, Assaf (39.3% and 49.5 for IMV and CAT06, respectively) showed better lambing rates than Churra (29.0% and 39.0%, respectively), and the CAT06 catheter showed significantly higher rates for each breed.

Development of extender based on soybean lecithin for its application in liquid ram semen.

The soybean lecithin is used as a phospholipids source for the commercial extenders available for freezing bull semen which allows replacing the traditional membrane protective of animal origin (egg yolk). These extenders have been tested for freezing semen in various livestock species but specific adjustments cannot be made due to trade protection. The aim of the present study was to develop a soybean-based extender analyzing the optimal conditions of preparation, handling, and storage in order to optimize its use in liquid ram semen. Its effect on the quality of liquid ram semen was also studied. Different TES-Tris-Fructose-based extenders were prepared using two soybean types (S20 and S95) differentiated by their lipid composition (complex or simple, respectively). These extenders were made up in two temperatures: 20 degrees C (PT20) or 37 degrees C (PT37); centrifuged and filtered at 20 degrees C and stored at 15 degrees C or 5 degrees C (ST15 and ST05) for several periods (from 6 hours to 7 days). Three different concentrations of soybean (0.5%, 2%, and 3.5%) were evaluated for each extender. The amount and nature of phospholipids present in the extender were evaluated by high performance liquid chromatography (HPLC) method according to the different parameters applied in their preparation. In general, the highest quantity of phospholipids is observed in S20 extender. Centrifugation-filtration process during the extender preparation reduces by 50% the quantity of phospholipids in medium for different experiments. The quantity of phospholipids was not affected significantly by preparation temperature in S20 extender. Storage temperature affects the phospholipids present in the extender (S20 and S95) with minimum values for the storage at 5 degrees C. As for the storage time, both extenders (S20 and S95) showed a stable quantity of phospholipids in the course of the time, for 2 days at 15 degrees C and for 7 days at 5 degrees C. The extender obtained with a higher concentration of soybean (3.5%) showed a higher content of phospholipids under different conditions tested. Finally, sperm motility and viability in new extenders were analyzed. We observed that the sperm quality is not affected by storage temperature for S20 extender. Sperm motility was higher in S20-2% extender and control (UL). Our results suggest that a soybean lecithin extender obtained from S20 soybean at 20 degrees C, centrifuged and filtered, preserve the sperm motility and viability at 15 degrees C and 5 degrees C as an egg-yolk extender.

Ovum pick-up in sheep: a comparison between different aspiration devices for optimal oocyte retrieval.

In vivo ovum pick-up (OPU) in sheep may be improved with a proper choice of aspiration elements (needle and tubing) and aspiration vacuum pressure. In the present study, two experiments were carried out. In Expt 1, visible follicles in ovaries of slaughtered ewes (treated separately according to their diameters: small<3 mm, medium 3-5 mm and large>5 mm) were aspirated using different combinations of the three studied factors such as aspiration flow rate (10, 20, 30, 40 and 50 ml water/min), needle gauge (18 and 20 G) and tubing inner diameter (1, 2 or 3 mm internal diameter). In Expt 2, a study with two 18 G needles of different lengths (18 G: 82 mm; 18 GL: 600 mm) was carried out, using ovaries obtained post-mortem, and performing in vivo laparoscopic follicular aspiration on ewes. We considered good quality oocytes as those with both complete compact cumulus and a homogeneous cytoplasm. Recovery rate, proportion of good quality oocytes (good quality oocytes/100 oocytes recovered) and overall efficiency (good quality oocytes/100 follicles aspirated) were noted. In Expt 1, aspiration flow rate affect remarkable proportion of good quality oocytes (69.5%, 50.5%, 44.8%, 36.5% and 28.3% for flows from 10 to 50 ml/min respectively, p<0.05). Needle gauge did not affect aspiration device efficiency. Thin and intermediate tubings were more effective (overall efficiency rates: 34.9%, 32.3% and 28.1% for 1, 2 and 3 mm respectively, p<0.05). Follicle size did not affect recovery rate, but proportion of good quality oocytes was higher for large (77.9%) and medium (64.4%) follicles (p<0.05). Finally, some combinations of the aspiration device showed greater effectiveness. In Expt 2, needle length did not influence recovery rate, but good quality oocytes rate was significantly modified both post-mortem and in vivo (good quality rate for 18 G vs 18 GL needles: 69.5% vs 47.7% and 58.1% vs 25.4%, post-mortem and in vivo respectively, p<0.05). We conclude that low-aspiration flow rates (10 and 20 ml/min) with thin or intermediate tubings (1 and 2 mm), and any short needle (18 G or 20 G) are the most adequate aspiration factors for OPU in sheep.

Effects of colchicine on the shape of chick neuroepithelial cells during neurulation.

We have analyzed the effects of colchicine on the cell shapes in chick neuroepithelium. Cell shapes were ascertained by the position of the nucleus in plastic serial sections. We tested three colchicine doses (5 X 10(-5) M, 5 X 10(-6) M, and 5 X 10(-7) M) by two experimental treatments (in ovo and in vitro). Colchicine treatment in vitro is always effective in depolymerizing microtubules of neuroepithelial cells and reduces the percentages of wedge-shaped cells in the median area of neuroepithelium. The same effect can be observed when the embryos are treated with 5 X 10(-5) M or 5 X 10(-6) M colchicine in ovo. A concentration of colchicine of 5 X 10(-7) M in ovo cannot disrupt microtubules in stage 8 and stage 10 embryos, and the percentage of wedge-shaped cells is the same as that of the untreated cells. In stage 6 embryos this colchicine dose effects the microtubules and the percentages of wedge-shaped cells. These facts are interpreted in respect to variations in microtubular resistance to microtubular-disrupting agents that are shown by the neuroepithelial cells from different developmental stages.

Fungiform papillae of the pig and the wild boar analyzed by scanning electron microscopy.

Fungiform papillae of wild boar and pig were studied by scanning electron microscopy (SEM). Four regions were defined on the tongue: rostral, medial and caudal thirds and lateral sides. Morphologically the fungiform papillae correspond with their denomination. Rostral and lateral tongue regions presented the largest average number of fungiform papillae. Taste pores opened onto the upper surface of the papillae and were easily identifiable by SEM. The total number of fungiform taste pores from both animals was the highest reported in the literature. The lateral papillae of wild boar and pig contained the largest average number of pores per papilla. This region must be important in taste sensitivity. Lateral and rostral papillae from both animals can provide a source of taste buds for study since each fungiform papilla presents numerous taste buds and these papillae are very abundant.

A study of the chick thymus microenvironment during development: analysis by monoclonal antibodies against thymic epithelium.

The process of T-lymphocyte differentiation within the thymus involves a series of molecular interactions. In this work we have carried out an analysis of the chick thymus microenvironment in order to evaluate its heterogeneity during development. We have produced 11 monoclonal antibodies whose staining patterns detected by the immunoperoxidase technique allowed us to divide them into five groups. A first group (E19-E2, P0-E5, and P15-T1) binds to thymic medullary stroma showing a reticular pattern on medullary epithelial cells and whose significance would be related to thymic stromal secretion. The second group of monoclonal antibodies (P15-T3) stains thymic corpuscles of 10- and 15-day chicks. The third group of antibodies includes P0-E1, P0-E3, P5-A6, and P15-T2 whose staining pattern is both medullary and cortical. The fourth group (P10-HB1 and P10-HB2) binds to thymic stromal and cortical thymocytes, and the fifth group (P5-A1) is characterized by the staining of medullary vessels of 5-day chicks. These five groups of monoclonal antibodies corroborate the existence of an antigenic diversity of the chick thymus microenvironment. Their possible relationships with T-cell differentiation and stromal-thymocyte interactions are discussed.

Stereological analysis of ultrastructural changes of surrounding tissues to neuroectoderm during chick embryo neurulation.

The cytometric evolution of different subcellular components in the epiblast and the mesoderm of chick embryo during neurulation has been studied with stereological methods. The coefficient of cellular form (CFc) has specific values for each type, the epiblast having a mean CFc of 0.474, while the mesoderm, which has ellipsoidal cells, has a mean CFc of 0.643. The nucleus does not show any change of its coefficient of form although its surface density increases significantly. The proportion of mitochondria, present in the cells of each embryonic layer, remains constant during the 4 stages, being higher in the mesoderm cells (epiblast 3.6%; mesoderm 4.3%). The epiblast cells show a stable vitelline reserve, though the relative proportions of lipid bodies and yolk droplets vary: the volume density of yolk droplets increases from stage 5 (3.1%) to stage 8 (7.7%), while the lipid bodies diminish from 7 to 3.6% during this period. The mesoderm cells undergo a remarkable loss of vitelline volume during the same period. The rough endoplasmic reticulum of each cellular type has a remarkable length density increase, the significance of which is discussed in relation to production of extracellular matrix.