Sward characteristics and performance of dairy cows in organic grass-legume pastures shaded by tropical trees.

The silvopastoral system (SPS) has been suggested to ensure sustainability in animal production systems in tropical ecosystems. The objective of this study was to evaluate pasture characteristics, herbage intake, grazing activity and milk yield of Holstein×Zebu cows managed in two grazing systems (treatments): SPS dominated by a graminaceous forage (Brachiaria decumbens) intercropped with different leguminous herbaceous forages (Stylosanthes spp., Pueraria phaseoloides and Calopogonium mucunoides) and legume trees (Acacia mangium, Gliricidia sepium and Leucaena leucocephala), and open pasture (OP) of B. decumbens intercropped only with Stylosanthes spp. Pastures were managed according to the rules for organic cattle production. The study was carried out by following a switch back format with 12 cows, 6 for each treatment, over 3 experimental years. Herbage mass was similar (P>0.05) for both treatments, supporting an average stocking rate of 1.23 AU/ha. Daily dry matter intake did not vary (P>0.05) between treatments (average of 11.3±1.02 kg/cow per day, corresponding to 2.23±0.2% BW). Milk yield was higher (P0.05) in subsequent years. The highest (P0.05) milk yields. Low persistence of Stylosanthes guianensis was observed over the experimental period, indicating that the persistence of forage legumes under grazing could be improved using adapted cultivars that have higher annual seed production. The SPS and a diversified botanical composition of the pasture using legume species mixed with grasses are recommended for organic milk production.

The argentophil reticular cells of the mammalian spleen. II. The argentophil reticular cell arrangement inside the red pulp and its relationship with the endothelial lining cells of the splenic sinuses.

The red pulp's argentophil reticular cell network of the spleen is composed by 3 types of fixed cells: 1. the primitive reticular cell, slightly argentophil; 2. the small reticular cell; 3. the larger reticular cell, strongly argentophil and phagocytic. This latter shows the classical morphological characteristics attributed to the reticular cells of the spleen. The large argentophil reticular cell may become free, constituting a 4th cell type, the free macrophage. A 5th reticular cell type is the dendritic cell found into the lymphatic follicles of the white pulp. The argentophil reticular cells of the red pulp assemble together to form the reticular cells' network, that occurs inside the red pulp cords. The primitive and the small reticular cell form the fundamental network on which the large cells are apposed. The reticular cells of this network maitain relationship with the arterial terminal vessels of the red pulp, being responsible by the ellipsoid structure. In those arteriolar segments without ellipsoid and in those mammalian species devoid of ellipsoid, the white pulp reticular cells, that surround the blood vessel as a part of the lymphoid periarteriolar sheath, mix with the red pulp's reticular cells and both can hardly be discriminated. The ellipsoids are formed by large argentophil cells arranged in concentrical layers around its lumen that sometimes appear devoid of endothelial lining cells. The red pulp's argentophil reticular cells, either the small or the large ones, contributed to the structure of the splenic sinuses' wall; its thin processes surround the sinus wall outside the endothelial lining cell as fibrillar structures that cross the back side of the lining cells. Two or more argentophil reticular cells send fibrillar processes to a single sinus. The perisinusal reticular cells may send a process between adjacent endothelial lining, cells that insinuate and attain the sinus lumen; this process becomes thick and eventually, the reticular cell enter the sinus lumen as a free macrophage. The argentophil reticular cells of the red pulp make connection between the capsule or the trabeculae and the reticular cell network. The endothelial lining cells of the splenic sinuses are not argentophil.

The blood circulation inside the spleen. I. The arterial blood vessels general distribution in the rat spleen.

As an attempt to investigate the different pathways followed by the blood into the spleen and to analyse their functional significance, a technique was used mainly based on the intraarterial perfusion of a Prussian blue "solution" added of some chemical mediators and vasoactive substances. Such technique provides results which may be analysed taking into account the effect of the anaesthetic used, that may influence the findings. From the anaesthetic used, the sulfuric ether and the barbital sodium produce vasoconstriction of the white pulp blood vessels, whereas the chlorpromazine-promethazine doesn't have this effect, and so the Prussian blue appears inside these vessels. The vasodilator drugs, such as succinonitrile and papaverine hydrochloride, show a general vasodilator effect on the splenic arterial system. Teh arterial vessels of the white and the red pulp, including those placed at the subcapsular areas, become enlarged; into the white pulp, either the central or the peripheral blood vessel plexus of the lymphatic follicle becomes evident. The latter readily constitutes the perifollicular and the pericolumnar plexus. The blood vessels of this plexus become permeable to the Prussian blue "solution" by the heparin sodium effect, and so the dye particles enter the marginal zone and the splenic sinuses. In addition, from the white pulp arteries arise 2 types of anastomotic arterioles which appear enlarged after succinonitrile treatment: The short anastomotic arterioles that crosses the marginal zone entering the red pulp near the white pulp; the long anastomotic arterioles which enter the red pulp and after a long course end up into or around a collector sinus. The addition of histamine dihydrochloride to the perfusion solutions shows a slight vasodilator effect mainly on the subcapsular penicillar arterioles, including the helicine arterioles. The adrenergic stimulation of the splenic blood vessels induces a generalized arterial constriction, except of the anastomotic arterioles, that becomes open; in such way, the blood pathway follows the course of the anastomotic arterioles and the collector arterioles also become constricted. The adrenergic vasoconstrictor effect is inhibited by the phenoxy-benzamine hydrochloride. The addition of acetylcholine chloride, in the dosage, used, induces a generalized arterial vessel constriction, mainly of the perifollicular plexus. This effect is inhibited by atropine sulfate which, on the other hand, produces evident enlargement of the perifollicular and pericolumnar arterial plexus.

A histochemical investigation on the percutaneous absorption of vitamin D synthesized into the mammal epidermis.

The vitamin D transepidermis absorption was studied by means of a histochemical technique suitable to detect this vitamin and to discriminate it from cholesterol and its esters. Such technique shows vitamin D inside the mast cell granules. As the mast cell granules contain metachromatic substances its own histochemical reactivity must be previously blocked by methylation. After this treatment the mast cell granules do not stain by toluidine blue and do not react to the peracetic acid-toluidine blue reaction. However, the granules remains reactive to alkaline permanganate-toluidine blue and to alkaline permanganate-Schiff reactions. These results show that the mast cell granules do not contain cholesterol but they contain vitamin D. The lack of cholesterol suggests that vitamin D is not synthesized inside the granules. As the mast cells may appears within the epidermis or in close relationship with the epidermis, although it is placed into the superficial dermis, it was admitted that the mast cells uptake vitamin D contained inside the epidermis intercellular compartment. In such instances, the vitamin D synthesized by the keratinocytes enter the intercellular compartment, where its synthesis accomplishes, and migrate towards the basement membrane. At the basal epidermis layer or after passing through the basement membrane the vitamin D is taken up by mast cells, where it is stored inside its granules.

A histochemical study on the vitamin D synthesis into the epidermis.

Using a suitable histochemical method vitamins D and 7-dehydrocholesterol could be shown into the epidermis of several mammal species. As the histochemical method used is able to discriminate vitamins D and 7-dehydrocholesterol from cholesterol and its esters, the sites where these vitamins were synthesized within the epidermis layers could be established. Vitamins D and 7-dehydrocholesterol were found into the epidermis in the same sites where cholesterol and its esters take place, such as: the keratinizing cell thick membrane and the stratum spinosum and stratum granulosum keratinocytes cytoplasm. Inside the keratinocyte cytoplasm vitamin D shows a granular pattern and appears weakly bound to proteins. The reactivity of such granules seems to be partially blocked as could be shown by an hydrolysis accomplished previously. After the hydrolysis reactive vitamin D was also found inside the epidermis intercellular space. The results suggest that vitamin D is synthesized into the cytoplasm of stratum spinosum and stratum granulosum keratinocytes, where it appears weakly bound to proteins. Afterwards it reaches the intercellular space, where its synthesis is accomplished and it becomes firmly protein-bound losing its histochemical reactivity. However, after a suitable hydrolysis the histochemical reactivity could be recovered. From the intercellular spaces vitamin D could take 2 fates: It was partially incorporated on the keratinizing cell thick membrane out surface and eliminated by means of the epidermis exfoliation. It was partially absorbed after passing across the basement membrane. On the other hand, the vitamin D placed inside the stratum spinosum and stratum granulosum keratinocytes cytoplasm become incorporated on the inner surface of the keratinizing cell thick membrane. The relationship between vitamin D biosynthesis and the epidermis lamellar bodies was discussed.

A histochemical method able to detect vitamins D and to discriminate it from cholesterol and its esters.

A suitable histochemical method able to discriminate vitamins D and 7-dehydrocholesterol from cholesterol and its esters is proposed taking into account the alkaline permanganate-toluidine blue reaction, the alkaline permanganate-Schiff reaction and the inhibitory effect of the HgCl2-formalin fixative. This method appears specific according to the results provided by spot test. On tissue sections the alkaline permanganate-toluidine blue reaction is more sensitive and more specific than the alkaline permanganate-Schiff reaction. A previous ribonuclease treatment or the methylation effect improves the specificity of the alkaline permanganate-toluidine blue reaction. The 2.4-dinitrophenylhydrazine blockade must be used previously to the alkaline permanganate-Schiff reaction as an attempt to improve its specificity. The previous hydrolysis carried on by some suitable treatments seems very useful to detect masked vitamins D and 7-dehydrocholesterol. The oxidized products accountable for the reactivity of vitamins D and 7-dehydrocholesterol to the alkaline permanganate-toluidine blue and alkaline permanganate-Schiff reactions were discussed.

A histochemical method suitable to discriminate free cholesterol from its esters and both from 7-dehydrocholesterol and vitamins D.

The peracetic acid-toluidine blue and the peracetic acid-Schiff reactions as well as the failure of HgCl2-formalin fixative for inhibiting these reactions appears as a very useful method for free cholesterol histochemical detection and to discriminate it from its esters and from some of its metabolic products such as 7-dehydrocholesterol and vitamins D. The cholesterol histochemical detection provided by those reactions appears specific taking into account the findings afforded by spot tests. The peracetic acid-toluidine blue reaction is very suitable for histochemical purposes on tissue sections, since it does not produces tissues damages, it is sensitive and the stained end product is almost insoluble in the solvents frequently used in histological techniques. The previous ribonuclease treatment and methylation are very useful in avoiding the basophil substances interference on the peracetic acid-toluidine blue reaction. The 2,4-dinitrophenylhydrazine carbonyl group blockade is useful to decrease the influence of carbonyl group on the peracetic acid-histological techniques. The previous ribonuclease treatment and methylation are very useful in avoiding the basophil substances interference on the peracetic acid-toluidine blue reaction. The 2,4-dinitrophenylhydrazine carbonyl group blockade is useful to decrease the influence of carbonyl group on the peracetic acid-histological techniques. The previous ribonuclease treatment and methylation are very useful in avoiding the basophil substances interference on the peracetic acid-toluidine blue reaction. The 2,4-dinitrophenylhydrazine carbonyl group blockade is useful to decrease the influence of carbonyl group on the peracetic acid-Schiff reaction. The peracetic acid-toluidine blue reaction is more sensitive and more specific than peracetic acid-Schiff reaction, since the tissues do contain Schiff reactive products unable to be 2,4-dinitrophenylhydrazine blocked which interfere on the results of the latter reaction decreasing its specificity and its sensitivity. The cholesterol histochemical detection based on the peracetic acid-toluidine blue and peracetic acid-Schiff reaction as it is suitable in discriminating free cholesterol from its esters as well as from some of its metabolic products it appears very useful for metabolic studies on tissue sections.

Histochemical aspects concerning the synthesis and the fate of cholesterol into the epidermis.

By means of a suitable histochemical method free cholesterol and its esters could be detected into the epidermis layers. The results show that in the stratum spinosum keratinocytes free cholesterol appears as an amorphous or granular structure apparently protein unbound; into the stratum granulosum keratinocytes the cholesterol becomes protein-bound and its most part undergoes esterified. The extracellular compartment nearly the stratum granulosum contains a little amount of cholesterol esters loosely bound to proteins. The results suggest that free cholesterol after being synthesized into the cytoplasm of the stratum spinosum and granulosum keratinocytes, it is partially esterified and becomes protein-bound, appearing as fine granules within the cytoplasm of the granulous cells. From this site it takes to fates:1. Its most part remains into the granulous cell cytoplasm and at the same time the granulous cell develop to the horny cell it is placed on the thick cell membrane inner surface contributing to its thickness; 2. Another part after reaching the extracellular compartment it is spread over the thick membrane out surface. Inside the thick cell membrane, into the horny layer, free cholesterol is continuously esterified since the keratinizing cell migrate to the periphery; however even at the most peripheral layers the free cholesterol predominates. Either free cholesterol or its esters, contained into the keratinizing cell thick membrane, were excreted throughout the horny layer exfoliation. The keratinizing cell cytoplasm does not contain neither free cholesterol nor its esters.

A technique to tocopherol (vitamin E) histochemical detection.

A technique to detect tocopherol histochemically was proposed in basis on the following schedule: 1. toluidine blue and Schiff reagent negativity before a suitable oxidation; 2. performic acid-toluidine blue and performic acid-Schiff reagent positivity after fixing in formalin-CaCl2; 3. performic acid-toluidine blue and performic acid-Schiff reagent negativity after fixing in formalin-HgCl2; 4. ferric ferricyanide reaction positivity not influenced by the formalin-HgCl2 blockade. This technique tested on filter paper strips loaded with several lipids, steroids, vitamins, proteins, amino acids and ribonucleic acid shows that it is specific to tocopherols among the tested substances. Used on tissue sections this technique appears as very suitable to histochemical purposes.

Identification of tocopherol (vitamin E) in the skin and its histochemical detection.

The epidermis upper layer reacts positively to the performic acid-toluidine blue and the performic acid-Schiff; such reactions were inhibited by a previous formalin-HgCl2 fixation. The substance accounted for that reactivity could be extracted from skin histological sections by means of the chloroform-methanol-HCl effect. If this same solvent was used on human skin shaven horny layer powder it was able to extract protein-lipid complex that displays the tocopherol histochemical reactivity. Since this complex was subjected to the KOH hydrolysis, an hexane-soluble lipid moiety could be isolated from the hexane-insoluble protein moiety. The isolated lipid moiety react as does the tocopherol histochemically, in opposite to the protein moiety that react negatively to the performic acid-toluidine blue and the performic acid-Schiff reactions. By using thin layer chromatography it was possible to isolate from the lipid moiety two substances identified as alpha-tocopherol and cholesterol in basis of its chromatographic properties, the first being the sole responsible by the performic acid-toluidine blue and performic acid-Schiff reactivity of the lipid moiety. From the other fractions isolated from the horny layer powder, either the lipid or the protein ones do not show positive performic acid-toluidine blue or performic acid-Schiff reaction. It was deduced, in basis of these findings that the positivity of such reactions on the epidermis upper layer keeps a close correlation with the alpha-tocopherol, contained by the protein-lipid complex. The ethylenic double bonds from lipids and the S-S group from protein could not be taken as responsible by them. On the other hand, the peracetic acid-toluidine blue reaction shows a proper histochemical meaning, since it is able to detect free cholesterol.

The spleen-hypophysis relationship - the effect of splenectomy on the young rat growth.

The spleen-hypophysis relationship particularly the effect of splenectomy on the body growth rate was studied on young rats from both sexes. The following experimental groups were tested: 1. sham-operated controls; 2. splenectomized rats; 3. marsupialized-spleen rats; 4. parabiosis rats; 5. parabiosis plus splenectomy from only one animal. The results were taking in basis on the regression lines of the body weight increasing on time, during 11 weeks. In addition, the hypophysis weight, the volume of its lobes and the adenohypophysis alpha, beta and cromophobe cells nuclear volume were estimated. The results show that splenectomy provides a significant increasing of body weight. The splenectomized rats show furthermore, a significant hypophysis weight increasing, a hypertrophy of its anterior lobe and morphological evidences of functional hyperactivity of its alpha-cells (estimated towards its nuclear volume increasing). The results suggest that the growth rate increasing of splenectomized young rats depends upon the adenohypophysis alpha-cells hypersecretion.

An analysis on the specificity of the histochemical techniques already proposed to detect catalases and peroxidases.

The spot test carried on filter paper strips appears as a very suitable technique to investigate the reactivity of catalases, peroxidases, porphyrins (bilirubin and protoporphyrin), metalloporphyrins (haemoglobin, haemin, haematin, chlorophyll and cyanocobalamin), ferric and ferrous salts. By using this technique the specificity of the already proposed techniques admitted as appropriate to detect histochemically peroxidases and catalases was investigated. The results shown from the already proposed techniques to detect peroxidases only the alpha-naphthol reaction is somewhat specific for this enzyme, if the results were taken immediately. However, if the results were taken after 24 h, the reaction loose all specificity. The other techniques proposed to detect peroxidase are not specific, either concerning the discrimination between catalases and peroxidases activity or regarding the possibility to differenciate an enzymic from a catalytic activity provided by haemic iron containing compounds and sometimes by iron salts. Our histochemical technique already proposed as suitable to detect catalases seams to be specific, since peroxidases do not react positively. By replacing benzidine for some others hydrogen donors the peroxidases histochemical techniques remain not specific and are unable to discriminate this enzyme from catalases. Porphyrins (protoporphyrin and bilirubin), magnesium and cobalt containing metalloporhyrins (chlorophyll and cyanocobalamin) do not produce oxidation of any hydrogen donors used. Iron salts are also able to give positive results with some techniques already proposed as suitable for peroxidases and catalases detection.

Immunochemical specificity of a benzidine technique proposed to catalases histochemical detection.

As an attempt to test the specificity of an histochemical technique proposed to detect catalases, an investigation was carried out by immunochemical techniques. Purified catalases were used after analysed immunochemically by double immune diffusion test and immunoelectrophoretic technique. These pure catalases induced, after injecting into guinea-pigs, anti-serums that react specifically with catalase and does not give any cross reaction with peroxidases and haemic iron containing compounds. By the direct and indirect immuno-fluorescence techniques it was shown an intense catalase reactivity inside the cytoplasm of adrenal cortex and hepatic cells, that appears as a granular pattern. These results are very similar to those provided by the histochemical technique, either concerning to the reactive cells or to the granular pattern of the positive reaction. In such instances, the immunochemical results suggest the specificity of the histochemical reaction. This specificity is confirmed by the previous treatment of tissue sections by catalases anti-serum. After this treatment either the immunochemical or the histochemical technique to detect catalases provide negative results on cells that before the treatment were strongly reactive.

Catalases and peroxidases histochemical detection; techniques suitable to discriminate these enzymes.

By using the benzidine reaction, on filter paper strips loaded with catalases, peroxidases, porphyrins, haemic iron compounds and iron salts, it was possible to establish 2 histochemical techniques able to detect and discriminate catalases and peroxidases. Spot test analytical studies show that only peroxidases oxidize benzidine in presence of a 0.0015 M H2O2 final concentration into the incubation medium. If a 0.0035 M H2O2 final concentration is used both peroxidases and haemic iron were able to oxidize benzidine. At a 0.01 M H2O2 final concentration the oxidative property of catalases become apparent and therefore at this H2O2 concentration either peroxidases or haemic iron, as well as catalases could be detected. By increasing the H2O2 concentration into the incubation medium, when a 4M concentration was chosen to detect histochemically catalases without any peroxidases interference. Using 0.0015 M and 4 M H2O2 final concentrations into the incubation medium it is possible to discriminate histochemically catalases and peroxidases. Several inhibitors of catalases and peroxidases were used as an attempt to try a specific inhibition of only one of these enzymes. It was demonstrated that the use of inhibitors does not help the histochemical discrimination between catalases and peroxidases.

Histochemical technique to detect choline-containing lipids.

An almost specific and highly sensitive technique to detect histochemically choline-containing lipids is proposed. This technique is based on the effect of Hg++ and phosphomolybdic ion on the choline, that yields insoluble complexes able to react with diphenylcarbazide producing a deep blue or violet stain. The specificity and the sensitivity of this technique was investigated on filter paper strips loaded with choline-containing lipids, choline-free lipids, steroids, vitamins, proteins or choline hydrochloride. The results show that the choline-containing substances react positively. The choline-free substances react negatively except calciferol and vitamin A that show weakly positive results. This technique used on tissue sections appears very suitable to histochemical purposes.