Recombinant Factors for Hemostasis

Trauma deaths are a result of hemorrhage in 37% of civilians and 47% military personnel and are the primary cause of death for individuals under 44 years of age. Current techniques used to treat hemorrhage are inadequate for severe bleeding. Preliminary research indicates that fibrin sealants (FS) alone or in combination with a dressing may be more effective; however, it has not been economically feasible for widespread use because of prohibitive costs related to procuring the proteins. To meet future demands for hemostatic therapies, FS will likely include recombinant human fibrinogen (rFI) and recombinant human Factor XIII (rFXIII). The underlying hypothesis of the research presented in this dissertation is that a liquid fibrin sealant (LFS) composed of recombinant FI, FXIII and FIIa in optimized proportions can assist hemostasis in the presence and absence of a bioresorbable bandage while using considerably fewer biologics than commercial products currently available. This dissertation characterized rFI produced in the milk of transgenic cows, plasma-derived thrombin (pdFIIa) activated by sodium citrate and rFXIIIa expressed in genetically engineered Pichia pastoris with respect to their capacity to serve as components in a LFS. The ratios of these factors were optimized to yield a LFS with a rapid clot formation rate and high viscoelastic strength. This optimized LFS was preliminarily tested ex vivo and in vivo. The clotting kinetics and viscoelastic strength of our optimized LFS was equivalent to those of a commercially available LFS; however, it uses approximately 75% less fibrinogen and thrombin. Our optimal LFS successfully achieved hemostasis in a significant number of the wounds that included extensive tissue and vascular damage. LFS applied without the assistance of a dressing was able to stop bleeding of oozing wounds or those with small vessels; however, a scaffold was needed when wounds contained large vasculature.

Análise multivariada de características reprodutivas e produtivas de vacas da raça Holandesa

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

PHOSPHITE AS PHOSPHORUS SOURCE TO GRAIN YIELD OF COMMON BEAN PLANTS GROWN IN SOILS UNDER LOW OR ADEQUATE PHOSPHATE AVAILABILITY

The effects of foliar and soil applied phosphite on grain yield in common bean (Phaseolus vulgaris L.) grown in a weathered soil under low and adequate phosphate availability were evaluated. In the first experiment, treatments were composed of a 2 x 7 + 2 factorial scheme, with 2 soil P levels supplied as phosphate (40 e 200 mg P dm(-3) soil), 7 soil P levels supplied as phosphite (0-100 mg P dm(-3) soil), and 2 additional treatments (without P supply in soil, and all P supplied as phosphite). In the second experiment, treatments were composed of a 2 x 3 x 2 factorial scheme, with 2 soil phosphate levels (40 e 200 mg P dm(-3) soil), combined with 3 nutrient sources applied via foliar sprays (potassium phosphite, potassium phosphate, and potassium chloride as a control), and 2 foliar application numbers (single and two application). Additional treatments showed that phosphite is not P source for common bean nutrition. Phosphite supply in soil increased the P content in shoot (at full physiological maturity stage) and grains, but at the same time considerably decreased grain yield, regardless of the soil phosphate availability. Foliar sprays of phosphite decreased grain yield in plants grown under low soil phosphate availability, but no effect was observed in plants grown under adequate soil phosphate availability. In general, foliar sprays of phosphate did not satisfactorily improve grain yield of the common bean plants grown under low soil phosphate availability.

Phosphite as phosphorus source to grain yield of common bean plants grown in soils under low or adequate phosphate availability

The effects of foliar and soil applied phosphite on grain yield in common bean (Phaseolus vulgaris L.) grown in a weathered soil under low and adequate phosphate availability were evaluated. In the first experiment, treatments were composed of a 2 x 7 + 2 factorial scheme, with 2 soil P levels supplied as phosphate (40 e 200 mg P dm-3 soil), 7 soil P levels supplied as phosphite (0-100 mg P dm-3 soil), and 2 additional treatments (without P supply in soil, and all P supplied as phosphite). In the second experiment, treatments were composed of a 2 x 3 x 2 factorial scheme, with 2 soil phosphate levels (40 e 200 mg P dm-3 soil), combined with 3 nutrient sources applied via foliar sprays (potassium phosphite, potassium phosphate, and potassium chloride as a control), and 2 foliar application numbers (single and two application). Additional treatments showed that phosphite is not P source for common bean nutrition. Phosphite supply in soil increased the P content in shoot (at full physiological maturity stage) and grains, but at the same time considerably decreased grain yield, regardless of the soil phosphate availability. Foliar sprays of phosphite decreased grain yield in plants grown under low soil phosphate availability, but no effect was observed in plants grown under adequate soil phosphate availability. In general, foliar sprays of phosphate did not satisfactorily improve grain yield of the common bean plants grown under low soil phosphate availability.

Dynamics of follicular growth and progesterone concentrations in cyclic and anestrous suckling Nelore cows (Bos indicus) treated with progesterone, equine chorionic gonadotropin, or temporary calf removal

The objective of this study was to investigate the effects of eCG and temporary calf removal (TCR) associated with progesterone (P4) treatment on the dynamics of follicular growth, CL size, and P4 concentrations in cyclic (n ¼ 36) and anestrous (n ¼ 30) Nelore cows. Cyclic (C) and anestrous (A) cows were divided into three groups. The control group received 2 mg of estradiol benzoate via intramuscular (IM) injection and an intravaginal device containing 1.9 g of P4 on Day 0. On Day 8, the device was removed, and the animals received 12.5 mg of dinoprost tromethamine IM. After 24 hours, the animals received 1 mg of estradiol benzoate IM. In the eCG group, cows received the same treatment described for the control group but also received 400 UI of eCG at the time of device removal. In the TCR group, calves were separated from the cows for 56 hours after device removal. Ultrasound exams were performed every 24 hours after device removal until the time of ovulation and 12 days after ovulation to measure the size of the CL. On the same day as the CL measurement, blood was collected to determine the plasma P4 level. Statistical analyses were performed with a significance level of P ≤ 0.05. In cyclic cows, the presence of the CL at the beginning of protocol resulted in a smaller follicle diameter at the time of device removal (7.4 ± 0.3 mm in cows with CL vs. 8.9 ± 0.4 mm in cows without CL; P ¼ 0.03). All cows ovulated within 72 hours after device removal. Anestrous cows treated with eCG or TCR showed follicle diameter at fixed-timed artificial insemination (A-eCG 10.2 ± 0.3 and A-TCR 10.3 ± 0.5 mm) and follicular growth rate (A-eCG 1.5 ± 0.2 and A-TCR 1.3 ± 0.1 mm/day) similar to cyclic cows (C-eCG 11.0 ± 0.6 and C-TCR 12.0 ± 0.5 mm) and (C-eCG 1.4 ± 0.2 and C-TCR 1.6 ± 0.2 mm/day, respectively; P ≤ 0.05). Despite the similarities in CL size, the average P4 concentration was higher in the A-TCR (9.6 ± 1.4 ng/mL) than in the A-control (4.0 ± 1.0 ng/mL) and C-TCR (4.4 ± 1.0 ng/mL) groups (P < 0.05). From these results, we conclude that eCG treatment and TCR improved the fertility of anestrous cows by providing follicular growth rates and size of dominant follicles similar to cyclic cows. Additionally, TCR increases the plasma concentrations of P4 in anestrous cows

Condição corporal ao parto e produção de leite sobre o desempenho reprodutivo de vacas holandesas em lactação

O objetivo, neste estudo, foi avaliar os efeitos da condição corporal ao parto e da mudança de condição corporal sobre o desempenho reprodutivo de vacas leiteiras após o parto. Para tanto, 51 vacas holandesas, de 30 dias pré-parto até 150 dias após o parto, foram distribuídas aleatoriamente de acordo com a condição corporal ao parto nas classes 1 (condição corporal maior ou igual a 3,25) e 2 (condição corporal menor ou igual a 3,0). Dentro das classes de condição corporal ao parto, os animais foram distribuídos quanto à mudança de condição corporal (Categoria 1, igual ou menor que -0,50 e Categoria 2, igual ou maior que -0,75) e à média da produção de leite ajustada para 3,5% aos 150 dias (Grupo 1 = 22,61 e Grupo 2 = 31,65 kg/dia). Não houve diferenças da condição corporal ao parto e da produção de leite ajustada para 3,5% sobre o intervalo parto primeiro estro, intervalo parto primeiro serviço, período de serviço, número de serviço / concepção e taxa de gestação aos 150 dias de lactação. Vacas que ao parto apresentaram média de escore de condição corporal de 3,40 e 2,79 tiveram semelhante desempenho reprodutivo pósparto. Em relação à produção de leite ajustada para 3,5%, vacas com produção média de 22,61 e 31,65 apresentaram similares resultados. A mudança de condição corporal não influenciou o intervalo parto primeiro serviço, o período de serviço e o número de serviço por concepção, mas apresentaram maior intervalo parto primeiro estro e menor taxa de gestação

Teat anatomy affects requirements for udder preparation in Mediterranean buffaloes

The present study was conducted to assess the interrelation between teat anatomy and machine milking in dairy buffaloes raised in Switzerland. A 3-min pre-stimulation induced milk ejection before cluster attachment in most cases and caused an optimal milk removal during machine milking. In an additional experiment, longitudinal cross-section ultrasound was obtained before and after a 3-min pre-stimulation. Teat wall thickness, teat diameter, cisternal diameter and teat canal length were evaluated. It was observed that 3-min pre-stimulation dramatically reduced teat canal length whereas all the other anatomical parameters remained unchanged. The vacuum needed to open the teat canal was also measured before and after a 3-min pre-stimulation by using a special teat cup with only the mouthpiece of the liner remaining on the top of the teat cup (no liner, no pulsation). Without pre-stimulation but after wetting the teat canal by stripping one squirt of milk out of the teat, no milk could be withdrawn with a vacuum up to 39 kPa. However, after pre-stimulation, milk flow occurred in all buffaloes at a vacuum between 16 and 38 kPa. In the last experiment, the teat tissue was examined in slaughtered buffaloes and compared with teat tissue of cows. No difference was noted in histological sections and teat canal length was similar in cows and buffaloes. Proximal to the teat canal, the teat did not pass into an open cistern but the lumen was collapsed. In conclusion, buffaloes need to be well pre-stimulated because the tissue above the teat canal provides additional teat closure before milk ejection. Therefore, milk can only be obtained after pre-stimulation.

Rumen-protected choline supplementation in periparturient dairy goats: effects on liver and mammary gland

The current study investigated the effects of supplementing rumen-protected choline (RPC) on metabolic profile, selected liver constituents and transcript levels of selected enzymes, transcription factors and nuclear receptors involved in mammary lipid metabolism in dairy goats. Eight healthy lactating goats were studied: four received no choline supplementation (CTR group) and four received 4g RPC chloride/day (RPC group). The treatment was administered individually starting 4 weeks before expected kidding and continuing for 4 weeks after parturition. In the first month of lactation, milk yield and composition were measured weekly. On days 7, 14, 21 and 27 of lactation, blood samples were collected and analysed for glucose, beta-hydroxybutyrate, non-esterified fatty acids and cholesterol. On day 28 of lactation, samples of liver and mammary gland tissue were obtained. Liver tissue was analysed for total lipid and DNA content; mammary tissue was analysed for transcripts of lipoprotein lipase (LPL), fatty acid synthase (FAS), sterol regulatory binding proteins 1 and 2, peroxisome proliferator-activated receptor gamma and liver X receptor alpha. Milk yield was very similar in the two groups, but R PC goats had lower (P < 0.05) plasma beta-hydroxybutyrate. The total lipid content of liver was unaffected (P = 0.890), but the total lipid/DNA ratio was lower (both P < 0.05) in RPC than CTR animals. Choline had no effect on the expression of the mammary gland transcripts involved in lipid metabolism. The current plasma and liver data indicate that choline has a positive effect on liver lipid metabolism, whereas it appears to have little effect on transcript levels in mammary gland of various proteins involved in lipid metabolism. Nevertheless, the current results were obtained from a limited number of animals, and choline requirement and function in lactating dairy ruminants deserve further investigation.

Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland

Different pathogens, such as Escherichia coli and Staphylococcus aureus, can be responsible for different outcomes of mastitis; that is, acute and severe or chronic and subclinical. These differences in the disease could be related to different mammary responses to the pathogens. The objective of this study was to determine if intramammary challenge with the endotoxins lipopolysaccharide (LPS), from E. coli, and lipoteichoic acid (LTA), from Staph. aureus, induce different immune responses in vivo in milk cells and mammary tissue. To provide a reference level for comparing the challenge and to show the different stimulation of the mammary immune system on a quantitatively similar level, dosages of LPS and LTA were chosen that induced an increase of somatic cells in milk to similar maxima. One udder quarter in each of 21 lactating dairy cows was challenged with 0.2 mug of LPS or 20 mug of LTA. From these quarters and from respective control quarters, milk cells or tissue biopsies were obtained at 0, 6, and 12h relative to the challenge to measure mRNA expression of tumor necrosis factor-alpha (TNFalpha), IL-1beta, IL-8, lactoferrin, and RANTES (regulated upon activation, normal T-cell expressed and secreted). Furthermore, if no biopsies were performed, hourly milk samples were taken for measurement of somatic cell count, lactate dehydrogenase (LDH), and TNFalpha. Somatic cell count increased in all treatments to similar maxima with LPS and LTA treatments. Concentrations of TNFalpha in milk increased with LPS but not with LTA. The activity of LDH in milk increased in both treatments and was more pronounced with LPS than with LTA. The mRNA expression of TNFalpha, IL-1beta, IL-8, and RANTES showed increases in milk cells, and LPS was a stronger inducer than LTA. Lactoferrin mRNA expression decreased in milk cells with LPS and LTA treatments. The measured factors did not change in either treatment in mammary tissue. Challenge of udder quarters with dosages of LPS and LTA that induce similar increases in SCC stimulate the appearance of different immune factor patterns. This dissimilar response to LPS and LTA may partly explain the different course and intensity of mastitis after infection with E. coli and Staph. aureus, respectively.

Alopecia areata in Eringer cows

Alopecia areata is a hair loss disorder in humans, dogs and horses with a suspected autoimmune aetiology targeting anagen hair follicles. Alopecia areata is only sporadically reported in cows. Recently, we observed several cases of suspected alopecia areata in Eringer cows. The aim of this study was to confirm the presumptive diagnosis of alopecia areata and to define the clinical phenotype and histopathological patterns, including characterization of the infiltrating inflammatory cells. Twenty Eringer cows with alopecia and 11 Eringer cows without skin problems were included in this study. Affected cows had either generalized or multifocal alopecia or hypotrichosis. The tail, forehead and distal extremities were usually spared. Punch biopsies were obtained from the centre and margin of alopecic lesions and normal haired skin. Histological examination revealed several alterations in anagen hair bulbs. These included peri- and intrabulbar lymphocytic infiltration, peribulbar fibrosis, degenerate matrix cells with clumped melanosomes and pigmentary incontinence. Mild lymphocytic infiltrative mural folliculitis was seen in the inferior segment and isthmus of the hair follicles. Hair shafts were often unpigmented and dysplastic. The large majority of infiltrating lymphocytes were CD3(+) T cells, whereas only occasional CD20(+) lymphocytes were present in the peribulbar infiltrate. Our findings confirm the diagnosis of T-cell-mediated alopecia areata in these cows. Alopecia areata appears to occur with increased frequency in the Eringer breed, but distinct predisposing factors could not be identified.

Binding sites of muscarinic and adrenergic receptors in gastrointestinal tissues of dairy cows suffering from left displacement of the abomasum

Muscarinic acetylcholine (M) and adrenergic (AR) receptors mediate gastrointestinal motility. Using radioligand binding assays and real-time polymerase chain reaction, the densities of binding sites and mRNA levels of M(2), M(3), alpha(2AD)- and beta(2)-AR were compared in muscle tissues from the abomasal fundus, pylorus, duodenum, caecum, and external loop of the spiral colon of eight cows with left displacement of abomasum (LDA), and of eight healthy cows. Specific binding of the [(3)H]-ligands to each of the four receptors was competitive and saturable. Binding sites of M(2) (all intestinal sites), M(3) (duodenum and caecum), and of alpha(2AD)-AR (abomasal fundus) were lower (P<0.05) in cows with LDA than in healthy cows. The coefficients of correlation between binding sites and mRNA transcripts of receptors were dissimilar in cows with LDA and healthy cows. The decrease in densities of M (intestine) and of alpha(2AD)-AR (abomasum) receptors suggests their implication in the impairment of motility associated with or leading to LDA.

Quantitative mRNA analysis of adrenergic receptor subtypes in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation

OBJECTIVE: To investigate the distribution of mRNA coding for 9 adrenoceptor subtypes in the intestines of healthy dairy cows and cows with cecal dilatationdislocation (CDD). SAMPLE POPULATION: Full-thickness specimens of the intestinal wall were obtained from the ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of 15 cows with CDD (group 1) and 15 healthy (control) cows (group 2, specimens collected during laparotomy; group 3, specimens collected after slaughter). PROCEDURES: Concentrations of mRNA for 9 adrenoceptor subtypes (alpha(1A), alpha(1B), alpha(1D), alpha(2AD), alpha(2B), alpha(2C), beta(1), beta(2), and beta(3)) were measured by quantitative real-time reverse transcriptase-PCR assay. Results were expressed relative to mRNA expression of a housekeeping gene. RESULTS: Expression of mRNA for alpha(1B)-, alpha(2AD)-, alpha(2B)-, beta(1)-, and beta(2)-adrenoceptors was significantly lower in cows with CDD than in control cows. In the ileum, these receptors all had lower mRNA expression in cows with CDD than in control cows. The same effect was detected in the ELSC for mRNA for alpha(2AD)-, alpha(2B)-, beta(1)-, and beta(2)-adrenoceptors, and in the cecum and PLAC for alpha(2B)- and beta(2)-adrenoceptors. Groups did not differ significantly for alpha(1A)-adrenoceptors. The mRNA expression for alpha(1D)-, alpha(2C)-, and beta(3)-adrenoceptors was extremely low in all groups. CONCLUSIONS AND CLINICAL RELEVANCE: Differences in expression of mRNA coding for adrenoceptors, most pronounced in the ileum and spiral colon, between cows with CDD and control cows support the hypothesis of an implication of adrenergic mechanisms in the pathogenesis of CDD in dairy cows.

Distribution of mRNA coding for 5-hydroxytryptamine receptor subtypes in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation

OBJECTIVE: To investigate the distribution of mRNA coding for 7 subtypes of 5-hydroxytryptamine receptors (5-HTRs) in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation (CDD). SAMPLE POPULATION: Full-thickness intestinal wall biopsy specimens were obtained from the ileum, cecum, proximal loop of the ascending colon, and external loop of the spiral colon (ELSC) of 15 cows with CDD (group 1) and 15 healthy dairy cows allocated to 2 control groups (specimens collected during routine laparotomy [group 2] or after cows were slaughtered [group 3]). PROCEDURE: Amounts of mRNA coding for 7 subtypes of 5-HTRs (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, and 5-HT4) were measured by quantitative real-time reverse transcriptase-PCR assay. Results were expressed as the percentage of mRNA expression of a housekeeping gene. RESULTS: Expression of mRNA coding for 5-HTR1B, 5-HTR2B, and 5-HTR4 was significantly lower in cows with CDD than in healthy cows. For 5-HTR2B and 5-HTR4, significant differences between cows with CDD and control cows were most pronounced for the ELSC. Expression of mRNA for 5-HTR1D, 5-HTR1F, and 5-HTR2A was extremely low in all groups, and mRNA for 5-HTR1A was not detected. CONCLUSIONS AND CLINICAL RELEVANCE: Relative concentrations of mRNA coding for 5-HTR1B, 5-HT2B, and 5-HTR4 were significantly lower in the intestines of cows with CDD than in the intestines of healthy dairy cows, especially for 5-HT2B and 5-HTR4 in the ELSC. This supports the hypothesis that serotonergic mechanisms, primarily in the spiral colon, are implicated in the pathogenesis of CDD.

Management, feeding, production, reproduction and udder health on organic and conventional Swiss dairy farms

Organic dairy farms (OP; n=60) and conventional dairy farms (integrated production, IP; n=60), matched in size, location, and agricultural zone (altitude), were studied for possible differences in management, feeding, production, reproduction and udder health. OP and IP farms were similar in size (17.7 and 16.9 ha), milk quota (65900 and 70,000 kg/year), cow number (14 and 15), cow age (5.3 and 5.2 years), housing of cows of the Simmental x Red Holstein or Holstein breeds (87 and 75%; 45 and 60%), but differed significantly with respect to loose housing systems (18 and 7%), outside paddocks (98 and 75%), energy-corrected 305-d milk yield (5,695 and 6,059 kg), milk protein content (31.8 and 32.7 g/kg), use of bucket milking systems (73 and 33%), observance of regular (12-h) milking intervals (47 and 68%), routine application of the California-Mastitis-Test (10 and 28%), teat dipping after milking (25 and 43%) and blanket dry cow treatments (0 and 45%). Milk somatic cell counts on OP and IP farms (119 000 and 117,000/mL) and reproduction data were similar and there were no significant differences between OP and IP farms as concerns available feeds, planning and management of feeding. Alternative veterinary treatments were used more often on OP than IP farms (55 and 17%). Main causes for cow replacements on OP and IP farms were fertility disorders (both 45%), age (40 and 42%), sale (30 and 37%) and udder health (35 and 13%).Between OP and IP Swiss dairy farms thus relatively few larger differences were found.

The diagnostic yield of vitrectomy specimen analysis in chronic idiopathic endogenous uveitis

PURPOSE: The low diagnostic yield of vitrectomy specimen analysis in chronic idiopathic uveitis (CIU) has been related to the complex nature of the underlying disease and to methodologic and tissue immanent factors in older studies. In an attempt to evaluate the impact of recently acquired analytic methods, the authors assessed the current diagnostic yield in CIU. METHODS: Retrospective analysis of consecutive vitrectomy specimens from patients with chronic endogenous uveitis (n = 56) in whom extensive systemic workup had not revealed a specific diagnosis (idiopathic) and medical treatment had not resulted in a satisfying clinical situation. Patients with acute postoperative endophthalmitis served a basis for methodologic comparison (Group 2; n = 21). RESULTS: In CIU, a specific diagnosis provided in 17.9% and a specific diagnosis excluded in 21.4%. In 60.7% the laboratory investigations were inconclusive. In postoperative endophthalmitis, microbiological culture established the infectious agent in 47.6%. In six of eight randomly selected cases, eubacterial PCR identified bacterial DNA confirming the culture results in three, remaining negative in two with a positive culture and being positive in three no growth specimens. A double negative result never occurred, suggesting a very high detection rate, when both tests were applied. CONCLUSIONS: The diagnostic yield of vitrectomy specimen analysis has not been improved by currently routinely applied methods in recent years in contrast to the significantly improved sensitivity of combined standardized culture and PCR analysis in endophthalmitis. Consequently, the low diagnostic yield in CIU has to be attributed to insufficient understanding of the underlying pathophysiologic mechanisms.