Analysis of the effect of temperature changes combined with different alkaline pH on the β-lactoglobulin trypsin hydrolysis pattern using MALDI-TOF-MS/MS

Temperature and pH influence the conformation of the whey protein β-lactoglobulin (β-Lg) monomer, dimer, and octamer formation, its denaturation, and solubility. Most hydrolyses have been reported at trypsin (EC 3.4.21.4) optimum conditions (pH 7.8 and 37 °C), while the hydrolysate mass spectrometry was largely limited to peptides with <4 kDa. There are few reports on trypsin peptide release patterns away from optimum. This work investigated the influence of alkaline (8.65 and 9.5) and optimum (7.8) pH at different temperatures (25, 37.5, and 50 °C) on β-Lg (7.5%, w/v) hydrolysis. Sample aliquots were drawn out before the addition of trypsin (blank sample) and at various time intervals (15 s to 10 min) thereafter. Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was used to monitor peptide evolution over time with the use of two matrixes: α-cyano-4-hydroxycinnamic acid (HCCA) and 2.5-dihydroxyacetophenone (DHAP). Mass analysis showed that the N- and C-terminals (Lys(8)-Gly(9), Lys(100)-Lys(101), Arg(124)-Thr(125), Lys(141)-Ala(142), and Arg(148)-Leu(149)) of β-Lg were cleaved early (15 s) implying the ease of trypsinolysis at the exposed terminals. Hydrolyses at 25 °C and pH 7.8 as well as at 50 °C and pH 9.5 were slowed down and ordered. Nonspecific chymotrypsin-like behavior occurred more at higher temperatures (50 °C) than at lower ones (25 and 37.5 °C). In addition to our earlier work in the acid pH region, it can be concluded that there is potential for controlled hydrolysis outside the trypsin optimum, where different target peptides with predictable biofunctionalities could be produced.     

Some features of coagulase positive staphylococci from bovine milk. II. Comparison of conventional techniques and the API Staph system

A total of 150 isolates of Staphylococcus aureus were subjected to the tests on the API Staph system. Of these, 50 were also tested by conventional methods, using the same tests as those found on the API Staph strips. Applying the principles of numerical taxonomy, the relationship between these isolates was 82% and more. Tests for the metabolism of sucrose and N-acetyl-glucosamine and for the production of argenine dihydrolysate and urease appear to be superfluous in the identification of S. aureus in this system.     

The occurrence of hypotaurine in ovarian follicle walls in sheep and in the Fallopian tube of sheep and other animals

Inhalt: In den Ovarien von Schafen kommen während des Brunstzyklus in den Folli-kelwandungen Hypotaurin und Inositol in Konzentrationen von 0–7 mg % bzw. 40 mg % vor. Hypotaurin wurde auβerdem in den Eileitern von Schaf, Pferd, Rind, Schwein und Kaninchen gefunden; in zwei Fällen gelang auch der Nachweis von Hypotaurin in der Fimbrienplatte der Hündin. Es wird die Hypothese aufgestellt, daβ bei mit Progesteron bebandelten Tieren das Fehlen von Hypotaurin möglicherweise die Entwicklung und den Transport der Eier ungünstig beeinfluβt.     

Some aspects of non-surgical embryo transfer in cattle

Non-surgical embryo recovery methods in cattle produce recovery rates similar to those obtained by surgical techniques. The average pregnancy rate following non-surgical transfers, (30%), is however much lower than that obtained following surgical transfer methods (60%).From the literature there is clear evidence of the existence of a luteolytic factor produced by the uterus and also some indication of a luteotropin which counteracts the luteolytic activity during pregnancy. Luteolysis of the corpus luteum is produced via the utero-ovarian pathway by the luteolytic factor present in the blood draining from most of the endometrium of the ipsilateral horn. The nature and mechanism of action of the luteotropic factor are not yet known but in order to counter the luteolytic effect, it must also exert its action in the ipsilateral horn.The unilateral relationship of the conceptus and the ovary bearing the corpus luteum explains the importance of embryo siting in determining the survival rate of embryos. After embryo deposition into the tip of the ipsilateral horn the antiluteolytic action of luteotropin is available either for local action or for conveyance to the corpus luteum. After embryo transfer to the contralateral horn or base of the ipsilateral horn the luteotropic factor has to travel via the uterine body down the ipsilateral horn in order to exert its influence. It is therefore concluded that embryos sited in the tip of the ipsilateral horn are more likely to survive than those at other locations which will require the transport of luteotropin produced in optimal quantities.Suggestions for further research to improve pregnancy rates following non-surgical methods are discussed.

---

Kurzfassung Die Anwendung nicht-chirurgischer Embryoerhaltungsverfahren bei Tieren führt zu Gewinnungsquoten, die denen von chirurgischen Verfahren entsprechen. Die durchschnittliche Trächtigkeitsquote von 30% nach nicht-chirurgischer Verlagerung ist viel niedriger als die bei chirurgischen Verlagerungsverfahren erreichten 60%.Die Fachliteratur hat die Existenz eines im Uterus erzeugten luteolytischen Faktors nachgewiesen und enthält einige Anhaltspunkte für ein Luteotropin, das der luteolytischen Tätigkeit während der Trächtigkeit entgegenwirkt. Die Luteolyse des Corpus luteum wickelt sich über den Uterus-Ovarial-Weg ab, über den das Blut mit dem luteolytischen Faktor aus dem Grossteil des Endometriums der ipsilateralen Tuba abläuft. Die Art des luteotropischen Faktors und der Mechanismus seiner Tätigkeit sind noch nicht bekannt; um jedoch die luteolytische Wirkung erfolgreich zu unterbinden, muss er seine Wirkung auch in der ipsilateralen Tuba ausüben, von der der luteolytische Faktor in das das Corpus luteum enthaltende Ovarium abgegeben wird.Die Einseitige Beziehung zwischen Fetus und Corpus luteum enthaltendem Ovarium erklärt die Bedeutung der Embryolagerung bei der Bestimmung der Überlebensrate von Embryos. Die antiluteolytische Wirkung von Luteotropin ist einzusetzen entweder als Wirkung an Ort und Stelle oder als Weiterleitung in das Corpus luteum, nach Embryoeinlagerung in die Spitze der ipsilateralen Tuba. Nach Embryoverlagerung in die kontralaterale Tuba, oder die Tubenbasis, muss der luteotropische Faktor über den Uteruskörper in die ipsilaterale Tuba hinunter verlagert werden, um eine entsprechende Wirkung auszuüben. Daher wird gefolgert, dass in der Spitze der ipsilateralen Tuba gelagerte Embryos die besten Überlebenschancen im Vergleich zu denjenigen in anderen Lagen haben, bei denen eine Weiterleitung des in optimalen Mengen erzeugten Luteotropins erforderlich ist.Anregungen für weitere Forschungstätigkeit zur Verbesserung der Trächtigkeitsquoten nach nicht-chirurgischen Verfahren werden erörtert.

---

Resume L'application des méthodes non-chirurgicales de récupération de l'embryon chez les bovins donne des taux de récupération analogues à ceux des techniques chirurgicales. Le taux moyen de gestation de 30% à la suite de transferts non-chirurgicaux est nettement inférieur aux 60% obtenus suivant les méthodes chirurgicales de transfert.La littérature établit clairement l'existence d'un facteur lutéolytique utérin et donne certaines indications de l'existence d'une lutéotropine qui contrarie l'activité lutéolytique pendant la gestation. La régression du corps jaune s'effecture via le canal utéro-ovarien qui draine le sang, contenant le facteur lutéolytique, de la plus grande partie de l'endomètre de la corne utérine homolatérale. La nature du facteur lutéotropique et le méchanisme de son action ne sont pas encore connus, mais pour que ce facteur neutralise l'effet lutéolytique, il faut qu'il exerce aussi son action dans la corne utérine homolatérale d'où le facteur lutéolytique est drainé vers l'ovaire contenant le corps jaune.La relation unilatérale du produit de la conception et de l'ovaire contenant le corps jaune explique l'importance de l'emplacement de l'embryon pour le taux de survie des embryons. L'action anti-lutéolytique de la lutéotropine s'exerce a l'emplacement de l'embryon soit localement soit par déplacement vers le corps jaune à la suite du dépôt de l'embryon au sommet de la corne utérine homolatérale.Après le transfert de l'embryon dans la corne contra-latérale ou base de la corne, le facteur lutéotropique doit être transporté via le corps utérin vers la corne utérine homolatérale pour exercer la même influence. La conclusion est donc que les embryons placés dans la pointe de la corne utérine homolatérale ont plus de chances de survie que ceux placés en d'autres endroits, et qui nécessiteront le transport de lutéotropine produite en quantités optimales.Des propositions de recherches complémentaires en vue d'améliorer les taux de gestation suivant des méthodes non-chirurgicales sont examineés.

---

Riassunto L'applicazione di metodi non chirurgici per il trapianto embrionale nei bovini si traduce in tassi di recupero simili a quelli ottenuti con tecniche chirugiche. Il tasso di gravidanza medio del 30% a seguito di trapianti incruenti è molto più basso del 60% che si ottiene con metodi di traslazione chirurgici.La letteratura dimostra chiaramente l'esistenza di un fattore luteolitico di produzione uterina; vi sono pure indicazioni della presenza di una luteotropina, che contrasta l'attività luteolitica durante la gravidanza. La luteolisi del corpo luteo ha luogo attraverso il dotto utero-ovarico, che permette il drenaggio del sangue contenente il fattore luteolitico da gran parte dell'endometrio del corno omolaterale. Natura e meccanismo d'azione del fattore luteotropico sono tuttora ignoti, ma se l'effetto luteolitico deve essere contrastato con successo, l'azione di tale fattore deve esplicarsi anche nel corno omolaterale, dal quale il fattore luteolitico viene drenato all'ovaia, che reca il corpo luteo.Il rapporto unilaterale fra concepito ed ovaia recante il corpo luteo spiega l'importanza della posizione dell'embrione per il tasso di sopravvivenza di quest'ultima. L'azione antiluteolitica della luteotropina si esplica sia localmente, sia per convogliamento al corno luteo dopo la deposizione dell'embrione nella punta del corno omolaterale. Dopo la spostamento dell'embrione al corno controlaterale, o alla base dello stesso, il fattore luteotropico deve essere convogliato tramite il corpo dell'utero al corno omolaterale onde esplicare analogamente la propria azione. Si conclude pertanto che gli embrioni situati nella punta del corno omolaterale hanno maggiori probabilità di sopravvivenza di quelli situati altrove, che abbisognano del convogliamento, in quantità ottimali, della luteotropina prodotta.Si discutono proposte di ulteriori ricerche tendenti a migliorare i tassi di gravidanza con l'applicazione di metodi non chirurgici.

     

Plasma amino acid concentrations in aggressive dogs

Following the hypothesis that metabolic screens may be useful tools in the diagnosis of canine aggression we have investigated the blood plasma amino acid levels of dogs which have been found aggressive (N = 10) against dogs or men in comparison to non-aggressive dogs (N = 10). In summary, the aggressive dogs showed elevated plasma concentrations of the neurophysiological active aromatic amino acids tryptophan (46/171 micromol/l, p < 0,001), tyrosine (38/67 micromol/l, p < 0.01) and histidine (74/91 micromol/l, p < 0.01) and lower lysine concentrations (175/151 micromol/l, p < 0.05), which seems to point to a stress situation of these dogs. The nitrogen metabolism is impaired in the urea-cycle in the conversion of ornithine (17/34 micromol/l, p < 0.01) to citrulline (64/47 micromol/l). Higher levels of branched chain amino acids, especially leucine (122/150 micromol/l, p < 0.01), mainly metabolized in muscles, and isoleucin (60/71 micromol/l, p < 0.05) show a high energy potential.The acidose-stimulator methionine (48/78 micromol/l, p < 0.01) proved elevated. The results show that the changed behavior in the aggressive dogs is also reflected in their free amino acid plasma concentrations, independent of the question whether these data are the cause or the result of the aggressivity.     

Renal cysts, nephrolithiasis, and ovarian cysts in a donkey suspected of having been sexually abused

A female donkey was thought by its owner to have been sexually abused because it had severe perineal swelling. Besides the perineal swelling and a very small vaginal erosion, there were no other abnormalities at clinical examination. Haematology and blood biochemistry revealed an increased leukocyte count, an elevated blood lactate concentration, and a low ionized calcium concentration. During night the donkey's condition deteriorated and it was euthanized in the morning. At necropsy severe haemorrhages were found within the subserosa of the caudal abdomen. Both kidneys were polycystic, and multiple calculi were found in the right kidney. Both ovaries had multiple cysts. Lesions (fibrosis and mineralization) were found in the liver, lungs, and mesenteric artery and were suggestive of an earlier parasitic infection. There was no evidence of sexual abuse.     

Detection of porcine circovirus genotypes 2a and 2b in aborted foetuses from infected swine herds in the State of S?o Paulo,Brazil

Background

Porcine circovirus type 2 (PCV2) has been associated with several disease complexes, including reproductive failure. The aim of this study was to identify the subtypes of PCV2 that are associated with reproductive failure in pigs from the State of São Paulo, Brazil and to investigate co-infections with other infectious organisms.

Findings

Samples of 168 aborted foetuses or mummified foetuses from five farrow-to-finish swine farms known to be infected with PCV2 and located in the State of São Paulo were tested for PCV2 by polymerase chain reaction (PCR). Positive samples were additionally tested for porcine parvovirus (PPV), Leptospira spp. and Brucella spp. by PCR. PCV2 was detected in 18 of the samples (10.7%). PPV, Brucella spp. and Leptospira spp were found in 2, 10 and 0 cases, respectively. Eleven PCV2 strains were sequenced and determined to be either genotype 2a (n = 1) or 2b (n = 10).

Conclusions

The findings indicate that the frequency of PCV2 infections in aborted porcine foetuses from the State of São Paulo is rather low (10.7%) and that co-infection with other pathogens is common and may be involved in PCV2 associated reproductive failure. No repeatable, characteristic amino acid motifs for regions of the PCV2 capsid protein seemed to be associated with abortion in sows.     

Leptospirosis as the most frequent infectious disease impairing productivity in small ruminants in Rio de Janeiro,Brazil

Despite the importance of small ruminants breeding in developing countries, milk/meat productivity remains unsatisfactory. Infectious diseases, such as leptospirosis, brucellosis, and small ruminant lentiviruses (SRLVs), contribute to this scenario. The objective of the present study was to determine the role of each of these diseases in the productivity of small ruminants breeding in Rio de Janeiro, Brazil. In goats, 343 samples were tested for leptospirosis, 560 for Brucella abortus, and 506 for caprine arthritis-encephalitis (CAE), whereas in sheep, 308 samples were tested for leptospirosis, 319 for B. abortus, 374 for Brucella ovis, and 278 for Maedi-Visna (MV). Regarding leptospirosis, 25.9% of goats and 47.4% sheep were seroreactive, with serovar Hardjo the most prevalent in both species. Anti-B. abortus agglutinins were found in 0.7% of all samples, exclusively in goats. In relation to SRLVs, 8.6% of goats and 3.2% of sheep samples were positive for CAE and MV, respectively. Leptospirosis was the major infectious problem in the small ruminants sampled and may contribute to impaired productivity of these animals.     

Effects of dietary carbohydrate sources on plasma glucose, insulin and IGF-I levels in multiparous sows

Effects of different carbohydrate sources on plasma glucose, insulin and insulin-like growth factor-I (IGF-I) levels were compared to subsequently be able to study effects of insulin-stimulating diets on follicle development in sows. The following feed components were tested in 12 sows during six consecutive test periods of 9.5 days: dextrose (DEX), sucrose (SUC), lactose (LAC), dextrose plus lactose (DL), sucrose plus lactose (SL), dextrose plus sugarbeet pulp (DSBP) and control (CON). On day 2, 5 and 9 of each test period, plasma glucose (only at day 9), insulin and IGF-I profiles were determined. Despite similar glucose profiles for all diets, the postprandial insulin response was higher for DL and SL compared with CON and LAC; the other diets were intermediate. Plasma IGF-I levels were higher in CON, LAC and SL compared with DSBP, but differences were only marginal. It was concluded that dextrose and sucrose have the potential to stimulate fast and high insulin peaks, especially when combined with additional lactose. Despite the high dextrose in the DSBP diet, the insulin response was flattened, probably due to the viscosity of sugarbeet pulp. The results show that modulation of plasma insulin levels by dietary carbohydrates seems possible in anabolic sows, but IGF-I levels are less easily modified.