Uterine leucocyte infiltration after artificial insemination in bitches

In the present study, polymorphonuclear neutrophils (PMN) were enumerated to evaluate acute uterine inflammation after artificial insemination in the bitch. It was concluded that the canine seminal plasma possessed an immunomodulating action. However, the most commonly used extender for freezing canine semen (Tris glucose with egg yolk and glycerol) was a potential inducer of uterine inflammation. (c) 2006 Published by Elsevier B.V.

Structural and functional characterization of myotoxin I, a Lys49 phospholipase A(2) homologue from Bothrops moojeni (Caissaca) snake venom

Myotoxin-I (MjTX-I) was purified to homogeneity from the venom of Bothrops moojeni by ion-exchange chromatography on CM-Sepharose. Its molecular weight, estimated by SDS-PAGE, was 13,400 (reduced) or 26,000 (unreduced). The extinction coefficient (E-1.0 cm(1.0 mg/ml)) of MjTX-I was 1.145 at lambda = 278 nm, pH 7.0, and its isoelectric point was 8.2 at ionic strength mu = 0.1. When lyophilized and stored at 4 degrees C, dimeric, trimeric, and pentameric forms of the protein were identified by SDS-PAGE. This heterogeneous sample could be separated into three fractions by gel filtration on Sephadex 6-50. The fractions were analyzed by isoelectric focusing, immunoelectrophoresis, and amino acid composition, which indicated that heterogeneity was the result of different levels of self-association. Protein sequencing indicated that MjTX-I is a Lys49 myotoxin and consists of 121 amino acids (M-r = 13,669), containing a high proportion of basic and hydrophobic residues. It shares a high degree of sequence identity with other Lys49 PLA(2)-like myotoxins, but shows a significantly lower identity with catalytically active Asp49 PLA(2)s. The three-dimensional structure of MjTX-I was modeled based on the crystal structures of three highly homologous Lys49 PLA(2)-like myotoxins. This model showed that the amino acid substitutions are conservative, and mainly the beta-wing region, and the C-terminal extended random coil. MjTX-I displays local myotoxic and edema-inducing activities in mice, and is lethal by intraperitoneal injection, with an LD50 value of 8.5 +/- 0.8 mg/kg, In addition, it is cytotoxic to myoblasts/ myotubes in culture, and disrupts negatively charged liposomes. In comparison with the freshly prepared dimeric sample, the more aggregated forms showed significantly reduced myotoxic activity. However, the edema-inducing activity of MjTX-I was independent of molecular association. Phospholipase A(2) activity on egg yolk, as well as anticoagulant activity, were undetectable both in the native and in the more associated forms. His, Tyr, and Trp residues of the toxin were chemically modified by specific reagents. Although the myotoxic and lethal activities of the modified toxins were reduced by these treatments, neither its edema-inducing or Liposome-disrupting activities were significantly altered. Rabbit antibodies to native MjTX-I cross-reacted with the chemically modified forms, and both the native and modified MjTX-I preparations were recognized by antibodies against the C-terminal region 115-129 of myotoxin II from B. asper, a highly Lys49 PLA(2)-homologue with high sequencial similarity. (C) 2000 Academic Press.

Assignment of the disulfide bridges in bothropstoxin-I, a myonecrotic Lys49 PLA(2) homolog from Bothrops jararacussu snake venom

Bothropstoxin-I (BthTX-1), a Lys49 phospholipase A(2) homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys50-Cys131, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-1 disulfide bonds follow the normal pattern of group II PLA(2)s.

Efeito de Diferentes Diluídores na Fertilidade de Éguas de Diferentes Haras Inseminadas com Sêmen Diluído, Resfriado e Transportado em Conteiner Especial

The effects of extender and farms on the fertility of sixty-five Mangalarga Marchador breed mares were studied. The mares were randomly assigned to a 2 x 2 factorial experiment, with two breeding farms (H1 and H2) and two extenders (glycine-egg yolk-T1 and lactose-egg yolk modified-T2), from only one stallion, with different number of replicates. The mares were inseminated, on alternated days, with diluted, cooled, transported semen, in a special container (MSP-2). There were no differences in the conception rate at first cycle, regardless of the breeding farm or extender used. Also. there was no difference among breeding farm or extender concerning the conception rate/cycle and the following reproductive characteristics: number of cycles/pregnancy, number of inseminations/mare, inseminations/pregnant mare, inseminations/open mare, water temperature in the container, temperature of semen in the container, time between collection and insemination and efficiency of pregnancy. The use of lactose-egg yolk modified extender for the dilution, cooling and transport of stallion semen between farms is recommended. The new container MSP-2 showed to be a option for the transportation between farms, for the same purpose.

Assignment of the bisulfide bridges in bothropstoxin-I, a Myonecrotic Lys49 PLA2 homolog from bothrops jararacussu snake venom

Bothropstoxin-I (BthTX-I), a Lys49 phospholipase A2 homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys50-Cysl31, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys 123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cysl05 by determination of the crystal structure, showing that BthTX-I disulfide bonds follow the normal pattern of group II PLA2s. © 2001 Plenum Publishing Corporation.

Avaliação do colesterol plasmático em coelhos com hipercolesterolemia induzida e tratados com extrato etanólico de própolis

The propolis (bee glue) is a product rich in flavonoids, which are known for antioxidant activities, a protective action to the lipoproteins LDL-cholesterol against lipid peroxidation. Because they have antioxidant properties, we investigated the effect of the ethanolic extract propolis on the plasma level of cholesterol in rabbits (Oryctolagus cuniculus) submitted to hypercholesterolaemia. The animals were divided into 4 groups. G 1=received commercial feed and water, G 2=received enriched feed and water, G 3=received enriched feed and ethanol, G 4=received enriched feed and ethanolic extract of propolis. The hypercholesterolaemia was induced with commercial feed enriched with egg yolk. The animals received the ethanolic extract propolis at the concentration of 100 mg/kg daily. Weekly, after fast of 14 hours, the samples of blood were collected from the marginal vein of the ear. The plasma was used for the estimation total cholesterol. From the results obtained, we verified that the ethanolic extract propolis significantly reduced the plasma level cholesterol (109,59 mg/dL, p<0,05), compared to the animals treated with ethanol (331,38 mg/dL), and also to those receiving the commercial feed only, with cholesterol at 269,74 mg/dL.

The ultrastructural aspects of vitellogenesis or oocyte secondary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Serrasalminae)

Oocyte secondary growth in S. spiloleura corresponds to the period in which different vesicular structures are formed, including the cortical alveoli and the yolk granules. The oocytes with cortical alveolus formation show vesicular structures with filamentous content in the cortical cytoplasmic region, which are the cortical alveolus precursors. In these oocytes, electron-dense vesicles of heterogenous content are dispersed in the inner cytoplasmic region and their nuclei are irregular, showing many nucleoli of different sizes. The oocytes in vitellogenesis are filled with many vesicles. The cortical alveolus precursors are in the peripheral region, and electron-dense granules are seen near to the nucleus. These fuse and form yolk granules. The oocytes in vitellogenesis show a very irregular nucleus that has nucleoli of different sizes. In the oocytes in final vitellogenesis, the yolk granules are scattered throughout the cytoplasm, displacing the cortical alveoli toward cell periphery. The nucleus is similar to the other stages.

High cholesterol intake modifies chylomicron metabolism in normolipidemic young men

Whether the consumption of egg yolk, which has a very high cholesterol content without excess saturated fats, has deleterious effects on lipid metabolism is controversial. Absorbed dietary cholesterol enters the bloodstream as chylomicrons, but the effects of regular consumption of large amounts of cholesterol on the metabolism of this lipoprotein have not been explored even though the accumulation of chylomicron remnants is associated with coronary artery disease (CAD). We investigated the effects of high dietary cholesterol on chylomicron metabolism in normolipidemic, healthy young men. The plasma kinetics of a chylomicron-like emulsion, doubly-labeled with 14C-cholesteryl ester ( 14C-CE) and 3H-triolein ( 3H-TG) were assessed in 25 men (17-22 y old, BMI 24.1 ± 3.4 kg/m 2). One group (n = 13) consumed 174 ± 41 mg cholesterol/d and no egg yolk. The other group (n = 12) consumed 3 whole eggs/d for a total cholesterol intake of 804 ± 40 mg/d. The nutritional composition of diets was the same for both groups, including total lipids and saturated fat, which comprised 25 and 7%, respectively, of energy intake. Serum LDL and HDL cholesterol and apoprotein B concentrations were higher in the group consuming the high-cholesterol diet (P < 0.05), but serum triacylglycerol, apo AI, and lipoprotein (a) did not differ between the 2 groups. The fractional clearance rate (FCR) of the 14C-CE emulsion, obtained by compartmental analysis, was 52% slower in the high-cholesterol than in the low-cholesterol group (P < 0.001); the 3H-TG FCR did not differ between the groups. Finally, we concluded that high cholesterol intakes increase the residence time of chylomicron remnants, as indicated by the 14C-CE kinetics, which may have undesirable effects related to the development of CAD. © 2006 American Society for Nutrition.

Studies on carbon-13 turnover in eggs and blood of commercial layers

This paper aimed at evaluating the influence of diets containing different isotopic values of carbon-13 turnover on the half-life of egg (yolk + albumen), yolk and albumen individually, and blood of poultry using δ‰ 13C isotopic variation. Commercial layers fed four experimental isocaloric and isonitrogenous diets (RC 4, RC 3, RMC 4 and RMC 3) containing different isotopic values, during an experimental period of 56 days. Turnover of the studied tissues was influenced by the experimental diets. Blood and albumen were more influenced by dietary treatments as compared to egg and yolk. The RMC 3 diet induced better performance (better feed intake and higher egg production) due faster rate of carbon substitution than the RC 4 diet, and lower half-life for egg (yolk + albumen), yolk, and albumen.

Cryopreservation of dog spermatozoa: Effect of bovine serum albumin on acrosomal integrity and pregnancy rates after artificial insemination

The objective of the present study was to compare the in vitro and in vivo profile of frozen dog semen with Tris-bovine serum albumin (TB) and Tris-egg yolk (TE) extenders. Twenty dogs were used as donors. Each dog was stimulated by penile massage and only the sperm-rich fraction was collected weekly until 40 ejaculates were obtained. After macroscopic and microscopic analysis, equal parts of each ejaculate were diluted with TB and TE by the one-step method at 37 °C. The semen was added to 0.5-mL French straws which presented normal characteristics before freezing and after thawing. Acrosomal integrity was evaluated by double Trypan blue-Giemsa staining, in which alive intact (LI), alive reacted (LR), dead intact (DI) and dead reacted (DR) spermatozoa, were identified by the time of thawing and up to 4 h of incubation at 39 °C, the TE being significantly superior to TB (P<0,01) in the LI and LR variables. The TB being significantly superior to TE (P<0,01) in the DR variable. Female dogs in natural heat were submitted to artificial insemination, 20 receiving TE-semen and 20 receiving TB-semen with the Osiris probe (IMV, L'Aigle, France) and the numbers indicate that TE was significantly better than TB (P<0,01) to pregnancy rate and number of puppies/delivery. We concluded from this study, that TE was better than TB, because this, induced an eady acrossome reaction in dog's sperm.

Efeito de um quelante de cálcio, um detergente e da lecitina de soja sobre a qualidade do sêmen caprino congelado-descongelado

The aim of this paper was verifying the effect of the Equex STM Paste and EDTA addition to a Tris-egg yolk extender, on the postthaw goat sperm viability. Nine semen samples of two adult goats were collected by artificial vagina and cryopreserved. It was also objective of this study, to evaluate the utilization of a soybean lecithin based commercial extender (Bioexcell® - IMV, L'Aigle, French) for the goat semen freezing. They were formed five experimental groups: TRIS; TRIS+EDTA; TRIS+EQUEX; TRIS+EDTA+EQUEX e Bioexcell. After evaluation, the semen was diluted in the five extenders and packed in 0.25mL straws with 100 million of motile spermatozoa. The samples were cooled at 0,46°C/min to 5°C, submitted at 75min of equilibration time and frozen in liquid nitrogen vapour. The thawing was accomplished in 37°C water bath for 50s. There were no differences (P>0,05) on the means of post-thaw total and progressive sperm motility among the groups TRIS, TRIS+EQUEX and TRIS+EQUEX+EDTA. The Bioexcell group obtained the least (P<0,05) percentage of post-thawing total and progressive sperm motility. After the thermotolerance test, it was observed the greatest (P<0,05) rates of total and progressive sperm motility in the Equex STM groups (TRIS+EQUEX and TRIS+EQUEX+EDTA). Thus, it can be affirmed that the Equex addition promotes better maintenance rates in the pos-thaw sperm viability, when compared with the extenders that did not contain it.

Congelação de espermatozoides epididimários de gatos utilizando o diluidor Botu-crio® após refrigeração por 24 h em contêiner de transporte de sêmen Botu-tainer®

The possibilities of using the sperm collected from the epididymis have been widely used because the fertilizing capacity sperm preservation and the possibility of using it for wild cats. But in the process of cryopreservation, some studies show a decrease in the quality of the sperm when left under cooling before frozen for some time. This study aimed to assess the quality of the epididymal sperm obtained from domestic cats after cryopreservation using a diluent based on egg yolk and glycerol (Botu-crio®), comparing the morphofunctional characteristics after cooling for 24 hours in a container of semen transport (Botu-tainer®). We use eight cats submitted to elective orchiectomy, aging from eight months, without racial determination, and good nutritional status. These sperm characteristics were: motility, vigor, concentration, membrane integrity and morphology. It has been found, after statistical analysis, that the container of semen was able to maintain sperm viability, even for 24h. We also observed a significant decrease on all parameters after frozen, consequential, probably to thermal stress that occurs in processing. However, the percentage of membrane integrity after thawing shows good employability of the Botu-crio®, which viability is possible to perform in vitro fertilization, requiring higher ratings.

Substitution of corn and soybean oil by cassava meal and African palm tree oil in diets of laying hens

Fourty-two White Leghorns laying hens, from the commercial Cuban hybrid L-33, were used for eight weeks during the laying peak (36 to 43 weeks of age), to assess the substitution of corn by cassava root meal (Manihot esculenta Crantz) and the crude soybean oil by crude oil of African palm tree (Elaeis guineensis J.) in the diets of laying hens. Analysis of variance was conducted, according to simple classification design, with three treatments and 14 repetitions (a cage with a hen). The treatments consisted of three diets (1- corn meal + soybean oil; 2- 25 % cassava meal + African palm tree oil; 3- 53 % cassava meal + African palm tree oil), with 15.71 % CP; 3.83 % Ca and 0.36 % P available. The viability was of 100 % in all treatments. No differences were found for laying (92.21, 92.09 and 91.59 %), which surpassed the potential of this hybrid during the laying peak (90 %), conversion (118g feedstuff/egg in the three treatments), egg mass produced (3066, 3114 and 3071 g/bird) and mass conversion (1.99, 1.95 y 1.98 feed consumed/egg mass). The pigmentation of the egg yolk was reduced as the level of cassava meal increased in the diets (6, 4 and 3 at Roche's scale), as well as the cost of the feed consumed in 56 d per hen (2.56, 2.15 and 1.83 USD/bird). The possibility of substituting, totally, corn meal by that of cassava and soybean oil by that of the African palm tree in the diets of laying hens during the laying peak was determined, with positive economic effect and without damaging the productive performance of birds.

Meal of cassava foliage as pigment of diets with cassava meal and oil of African oil palm for laying hens

Twenty-eight White Leghorn laying hens, of the Cuban commercial hybrid L-33, were used for eight weeks within the laying peak (36 to 43 weeks of age), to evaluate the meal of root of cassava (Manihot esculenta Crantz) and the crude oil of African oil palm (Elaeis guineensis J.) as pigments of the egg yolks, out of their incorporation to the diet of laying hens during the laying peak. The birds were allocated in individual cages, with 108 g of feed/bird/d, water ad libitum, and 16 h of light. Analysis of variance was performed, according to one-way design with two treatments and fourteen repetitions (one cage with one hen). The treatments consisted of two diets (I- cassava meal + African palm oil, II- cassava meal + African oil palm + 2.5 % of cassava foliage meal). The viability was of 100 % in all the treatments. No differences were found for laying (91.59 and 90.10 %), which surpassed the potential of this hybrid during the laying peak (90 %), feed conversion/egg (118 and 120 g of feed/egg), mass of egg produced (3071 and 3027 g/bird) and mass conversion (1.98 and 2.00). The pigmentation of the egg yolk was doubled, by adding 2.5 % of meal of cassava foliage to the diets (3 and 6 in the scale of Roche). It was likeable to enhance the pigmentation of the egg yolk and reduce the feeding costs of the laying hens, when including 2.5 % of meal of cassava foliage to diets where the cassava meal and the oil of the African oil palm are basic sources of starch and lipids for the laying hens.