Comparative stability of canine and feline hemostatic proteins in freeze‐thaw‐cycled fresh frozen plasma

Objective – To evaluate the stability of canine and feline hemostatic proteins in freeze‐thaw‐cycled (FTC) fresh frozen plasma (FFP). Design – Prospective study. Setting – Veterinary Teaching Hospital. Animals – Nine blood donor dogs and 10 blood donor cats. Interventions – Whole blood was collected and separated into packed RBC and plasma units according to standard methods. Each unit of plasma was divided into 2 equal aliquots and frozen (?41°C). One aliquot from each donor (FTC) was then thawed and then refrozen (?41°C) until time of analysis. The second aliquot (nonfreeze‐thaw‐cycled; NFTC) remained frozen until time of analysis. The hemostatic proteins assessed included coagulation factors, anticoagulant factors (antithrombin and Protein C), and adhesive proteins (fibrinogen and von Willebrand Factor). The coagulant activities of factors II, VII, VIII, IX, X, XI, and XII were measured in modified one‐stage activated partial thromboplastin time or prothrombin time assays. Antithrombin and Protein C activities were measured in chromogenic substrate assays. Clottable fibrinogen was measured via the Clauss method, and von Willebrand Factor concentration (vWF:Ag) was measured in an ELISA. A paired t‐test was utilized to identify differences in factor activity or concentration between FTC FFP and NFTC FFP. Measurements and Main results – No clinically or statistically significant differences (all P>0.05) were identified between FTC FFP and NFTC FFP. Conclusions – Refreezing FFP within 1 hour of initial thawing appeared to have no deleterious effects on the hemostatic protein activity or content of that unit. Transfusion of FTC FFP is expected to provide the recipient with comparable replacement of hemostatic proteins as FFP that has remained frozen.     

Comparative observation of freeze–thaw‐induced damage in pig,rabbit, and human corneal stroma

Objective Although variations exist between species with respect to outcomes after cryopreservation, little is known about the differences in the susceptibility of the corneal stroma to cryoinjury. We performed this study to investigate freeze–thaw‐induced damage in keratocytes and collagen in rabbit, pig, and human corneas. Animals studied Rabbit, pig, and human. Procedures We prepared 250‐μm‐thick anterior stroma from rabbit, pig, and human corneas after scraping off the epithelium and endothelium. Each 250‐μm‐thick corneal stroma without epithelium was placed in a 50‐mL tube, frozen with liquid N₂ for 15 min and taken out to thaw rapidly at 37 °C. This procedure of rapid freezing and thawing was repeated three times. Differences between the species with respect to cells and collagen structures were examined using hematoxylin–eosin (H&E) staining, terminal deoxynucleotidyl transferase‐mediated nick end labeling (TUNEL) assay, and transmission electron microscopy (TEM). We orthotopically transplanted the pig and rabbit corneal transplants after the triple freeze–thaw cycle into rabbit eyes and evaluated graft survival. Results On gross examination, rabbit corneas became opaque after the triple freeze–thaw procedure, while pig and human corneas remained transparent. Histologically, keratocytes were apoptotic on TUNEL assay and TEM in rabbit, pig, and human corneas. Collagen fibrils were fragmented and the arrangement of collagen fibrils was severely disturbed in rabbit corneas on H&E staining and TEM; collagen was well preserved in pig and human corneas. Rabbit corneal stroma underwent autolysis after transplantation, whereas the pig corneal stroma remained clear for 1 month. Conclusions Our study showed that rabbit corneal stroma was more susceptible to freeze–thaw injury than pig and human corneas.     

Immunohistochemical study of matrix metalloproteinases‐2 and ‐9, macrophage inflammatory protein‐2 and tissue inhibitors of matrix metalloproteinases‐1 and ‐2 in normal,purulonecrotic and fungal infected equine corneas

Objective Determine the effects of matrix metalloproteinases (MMPs)‐2, ‐9, macrophage inflammatory protein‐2 (MIP‐2), tissue inhibitors of matrix metalloproteinase (TIMP)‐1 and ‐2 by immunohistochemical expression in fungal affected and purulonecrotic corneas. Procedure Paraffin‐embedded equine corneal samples; normal (n = 9), fungal affected (FA; n = 26), and purulonecrotic without fungi (PN; n = 41) were evaluated immunohistochemically for MMP‐2, ‐9, MIP‐2, TIMP‐1 and ‐2. The number of immunoreactive inflammatory cells was counted and statistics analyzed. Western blot was performed to detect MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 proteins. Results Matrix metalloproteinases‐2, ‐9, MIP‐2, TIMP‐1 and ‐2 immunoreactivity was identified in corneal epithelium of normal corneas, and in corneal epithelium, inflammatory cells, keratocytes, and vascular endothelial cells of both FA and PN samples. Inflammatory cell immunoreactivity was significantly higher in FA and PN samples than in the normal corneas. There was positive correlation between MMP‐2 and MIP‐2, MMP‐9 and MIP‐2, and MMP‐9 and TIMP‐1 in inflammatory cell immunoreactivity in FA samples. There was positive correlation between MMP‐9 and MIP‐2, MMP‐9 and TIMP‐2, MIP‐2 and TIMP‐1, and MIP‐2 and TIMP‐2 in inflammatory cell immunoreactivity in PN samples. Western blot confirmed the presence of all four proteins in equine corneal samples. Conclusion Increased immunoreactivity of MMP‐2 and ‐9 in FA and PN samples is indirectly related to MIP‐2 through its role in neutrophil chemo‐attraction. Tissue inhibitors of matrix metalloproteinase‐1 and TIMP‐2 are up‐regulated in equine purulonecrotic and fungal keratitis secondary to MMP‐2 and MMP‐9 expression. The correlation between MMPs ‐2 and ‐9, MIP‐2, TIMPs ‐1 and ‐2 suggests that these proteins play a specific role in the pathogenesis of equine fungal keratitis.     

Feeding of grey squirrels (Sciurus carolinensis) with the contraceptive agent DiazaCon™: effect on cholesterol,hematology, and blood chemistry

Grey squirrels (Sciurus carolinensis) are an invasive species in Britain and Italy. They have replaced native red squirrels (Sciurus vulgaris) throughout most of Britain, and cause damage to trees. Currently, lethal control is used to manage grey squirrel populations in Britain, but nonlethal methods might be more acceptable to the public. One such method is contraception with 20,25‐diazacholesterol dihydrochloride (DiazaCon?). DiazaCon? inhibits the conversion of desmosterol to cholesterol, resulting in increasing desmosterol concentrations and decreasing cholesterol concentrations. Because cholesterol is needed for the synthesis of steroid reproductive hormones, such as progesterone and testosterone, inhibition of cholesterol synthesis indirectly inhibits reproduction. Desmosterol is used as a marker of efficacy in laboratory studies with species that do not reproduce readily in captivity. Grey squirrels were gavaged with a DiazaCon? solution for 2 days, and then fed DiazaCon?‐coated peanuts for an additional 8 days at target doses of 50 and 100 mg DiazaCon? per kg body weight. There was a significant difference in cholesterol concentrations in the treatment groups compared to the control group. Cholesterol was reduced by ≥ 40% for 2 months in both treatment groups. There were no differences among groups with respect to blood chemistry and hematology parameters, and mean values are reported. The mean overall dose of DiazaCon? received was 29.0 ± 1.6 and 55.3 ± 4.3 mg/kg in the low (50 mg/kg) and high dose (100 mg/kg) groups, respectively. DiazaCon? might provide an effective, acceptable alternative to lethal control.     

Long‐term feeding effects of dietary protein levels on egg production,immunocompetence and plasma amino acids of laying hens in subtropical condition

The objective of this study was to determine the effect of protein levels on production performance, immune response, liver triglyceride and plasma amino acids profile of laying hens during 21–48 weeks of age. Two groups of commercial hens (Babcock B‐308) were fed with diets with different crude protein levels (14% and 18% CP). The high protein diet (18% CP) significantly increased production performance, essential amino acid intake, spleen weight (% of body weight), ND‐titre (haemagglutination‐inhibition test), serum albumin (g/dl), serum alpha‐globulin and plasma essential amino acids (except histidine) and decreased liver triglyceride (mg/g liver) in relation to the low protein diet (14% CP). In long‐term effect, therefore, this study suggested that adequate dietary condition of amino acids, particularly methionine and branch‐chain amino acids is necessary for sustaining normal immunocompetence and achieving maximum production performance.     

A comparative study of the effects of intramuscular administration of gonadorelin,mating and intrauterine infusion of either raw seminal plasma or seminal plasma purified β‐NGF on luteal development in llamas

The aim of this study was to compare the effect of the intramuscular administration of 50 μg of gonadorelin acetate versus natural mating, intrauterine infusion (i.u.) of a physiological relevant dose of either raw llama seminal plasma (SP) or purified beta‐nerve growth factor from seminal origin (spβ‐NGF) on ovulation rate and corpus luteum (CL) development and function in llamas. Females with a follicle (≥8 mm) were assigned to groups: (i) i.m. administration of 50 μg of gonadorelin acetate (GnRH; positive control; n = 4); (ii) single mating (mating; n = 6); (iii) i.u. infusion of 4 ml of llama SP (SP; n = 4); or (iv) i.u. infusion of 10 mg of spβ‐NGF contained in 4 ml of PBS (phosphate‐buffered saline) (spβ‐NGF; n = 6). Ovaries were examined by power Doppler ultrasonography at 0, 1, 3, 6, 12 and 24 hr after treatment to determine preovulatory follicle vascularization area (VA), and additionally every 12 hr until Day 2 (Day of treatment = Day 0) to determine ovulation. Afterwards, ovaries were examined every other day until Day 8 to evaluate CL diameter and VA. Blood samples were collected on Days 0, 2, 4, 6 and 8 to determine plasma progesterone (P4) concentration. Ovulation rate did not differ (p = .7) among groups, but treatment affected (p < .0001) preovulatory follicle VA. Neither treatment administration nor treatment by time interaction affected (p ≥ .4) CL diameter, VA and plasma P4 concentration. Mating tended (p = .08) to increase CL VA when compared to the seminal plasma group by Day 8. Intrauterine administration of seminal plasma or spβ‐NGF does not increase CL size and function when compared to i.m. GnRH treatment, suggesting that the administration route of spβ‐NGF influences its luteotrophic effect in llamas.     

Effect of estradiol‐17β during in vitro growth culture on the growth,maturation, cumulus expansion and development of porcine oocytes from early antral follicles

Growing porcine oocytes from early antral follicles can acquire meiotic and developmental competence under suitable culture conditions, but at lower rates compared to full‐grown oocytes. We postulated that estradiol‐17β (E₂) supported the acquisition of meiotic and developmental competence as well as cumulus‐expansion ability during growth culture. Growing oocytes from early antral follicles (1.2 to 1.5 mm in diameter) were grown in vitro for 5 days in a medium containing 0, 10^?7, 10^?6, 10^?5 or 10^?4 mol/L E₂; after in vitro maturation, 35, 58, 47, 74 and 49% of oocytes matured to metaphase II, 25, 79, 77, 90 and 97% acquired cumulus‐expansion ability, and 23, 54, 63, 89 and 64% were fully surrounded by cumulus cells, respectively. Following maturation, electro‐stimulation was applied to the oocytes grown with 10^?5 mol/L E₂. After 6 days of culture, in vitro‐grown oocytes developed to the blastocyst stage at a rate similar to that for full‐grown oocytes (31% and 40%, respectively). Therefore, we suggest that the use of E₂ during growth culture improves the meiotic and developmental competence of oocytes, cumulus‐expansion ability, and cumulus cell attachment to the oocytes.