Phosphofructokinase and Malate Dehydrogenase Participate in the In Vitro Maturation of Porcine Oocytes

Oocyte maturation depends on the metabolic activity of cumulus–oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2‐oxoglutarate (5, 10 and 20 mm ) or hydroxymalonate (30, 60 and 100 mm ) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10^?5 and (2.54 ± 0.32) 10^?5, and for MDH, the U were (4.72 ± 0.42) 10^?5 and (4.38 ± 0.25) 10^?5 for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10^?3 and (0.94 ± 0.12) 10^?3, and for MDH (9.08 ± 0.93) 10^?3 and (1.89 ± 0.10) 10^?3 for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2‐oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.     

Immunohistochemical Localization of RANK, RANKL and OPG in Healthy and Arthritic Canine Elbow Joints

Objective— To determine if the receptor activator of nuclear factor-κB–receptor activator of nuclear factor-κB ligand–osteoprotegerin (RANK–RANKL–OPG) system is active in bone remodeling in dogs and, if so, whether differences in expression of these mediators occur in healthy and arthritic joints.
Study Design— Experimental study.
Sample Population— Fragmented processus coronoidei (n=20) were surgically removed from dogs with elbow arthritis and 5 corresponding healthy samples from dogs euthanatized for reasons other than elbow joint disease.
Methods— Bright-field immunohistochemistry and high-resolution fluorescence microscopy were used to investigate the distribution of RANK, RANKL, and OPG in healthy and arthritic joints.
Results— All 3 molecules were identified by immunostaining of canine bone tissue. In elbow dysplasia, the number of RANK-positive osteoclasts was increased. In their vicinity, cells expressing RANKL, a mediator of osteoclast activation, were abundant whereas the number of osteoblasts having the potential to limit osteoclastogenesis and bone resorption via OPG was few.
Conclusions— The RANK–RANKL–OPG system is active in bone remodeling in dogs. In elbow dysplasia, a surplus of molecules promoting osteoclastogenesis was evident and is indicative of an imbalance between the mediators regulating bone resorption and bone formation. Both OPG and neutralizing antibodies against RANKL have the potential to counterbalance bone resorption.
Clinical Relevance— Therapeutic use of neutralizing antibodies against RANKL to inhibit osteoclast activation warrants further investigation.     

The dispersal of Culicoides brevitarsis in eastern New South Wales and associations with the occurrences of arbovirus infections in cattle

Distributions of the vector Culicoides brevitarsis Kieffer (Diptera: Ceratopogonidae) (determined from light trap data) and 2 arboviruses (determined from seroconversions in sentinel cattle) were studied in eastern New South Wales in 1993–94. C brevitarsis was recorded progressively from endemic areas on the north coast, to Nowra on the south coast, and westward to Scone, in the Hunter Valley. C brevitarsis also survived through winter at Paterson, in the Hunter Valley. Its apparently focal reappearance in this marginal area had no obvious effect on the broad pattern of its progression or the dispersal of Akabane and bluetongue viruses. These viruses were first recorded from foci near Coffs Harbour, on the mid-north coast. Their first occurrences at different locations were associated with those of C brevitarsis, but not with each other. The viruses were found only within the recorded limits of the vector's distribution. Delays between the initial occurrence of C brevitarsis and first evidence of virus transmissions at locations ranged from 2 to 7 months. The delays decreased away from the points of focus and were negatively associated with the time of initial occurrence of the vector. Seroconversions to the viruses were related to the presence of C brevitarsis. However, the densities of C brevitarsis had no apparent effect on the initial numbers of cattle seroconverting to either virus. The results support the conclusion that the progressions of C brevitarsis and Akabane and bluetongue viruses were the result of gradual movements by the vector.     

The Effect of Storage on Ammonia Concentration in Canine Packed Red Blood Cells.

Objective: To determine the effect of storage on ammonia concentration in canine packed red blood cell (pRBC) units.
Design: In vitro and in vivo study.
Setting: University Veterinary Teaching Hospital.
Interventions: Ammonia concentration was measured in 7 units of canine pRBC prepared in citrate-phosphate-dextrose (CPD) and Adsol^a on Days 1 and 35 of storage. Ammonia was measured in 4 additional units of canine pRBC on Days 0, 7, 14, 21, 28, and 35. Plasma ammonia was also determined in 5 anemic dogs receiving pRBC.
Measurements and Main Results: Ammonia concentration increased from 73 ± 15 mmol/L (mean ± SD) on Day 1 to 800 ± 275 mmpl/L on Day (p<0.001). When measured every 7 days in 4 units of canine pRBC, ammonia concentration increased from 23 ± 8 mmol/L on Day 0 to 179 ± 13 mmol/L (Day 7), 276 ± 56 mmol/L (Day 14). 383 ± 47 mmol/L (Day21), 466 ± 30 mmol/L (Day 28), and 562 ± 27 mmol/L (Day 35) (p<0.05 for all comparisons). In a preliminary study, plasma ammonia concentration measured in blood samples from 5 anemic dogs without primary liver disease immediately before and after transfusion with 5–10 ml/kg of stored pRBC remained in the normal reference range.
Conclusions: The ammonia concentration in stored canine pRBC increased markedly with time. In this preliminary study, ammonia concentrations in dogs without primary liver disease did not increase above the reference range after transfusion with pRBC.     

Impact of storage conditions on the apple allergen Mal?d?1

Consumption of fresh apple fruits can induce allergic reactions in humans. The apple allergen Mal?d?1 is responsible for these allergic reactions in humans in Central Europe and North America. Biosynthesis of Mal?d?1 depends on apple cultivar, and its concentration increases with time during fruit storage. However, data on the impact of different fruit storage conditions during long-term storage are scarce. Hence, the Mal?d?1 contents of eight apple cultivars were analyzed for this study during long-term storage in a cold chamber as well as under controlled atmosphere conditions (CA). After harvest, apple fruits were stored for 12, 20, 28 or 36 weeks in a cold-chamber at +?2?°C or as under controlled atmosphere conditions of 1.5?% CO₂, 1.5?% O₂ at +?2?°C. Mal?d?1 content in apple fruit of all eight cultivars examined increased during fruit storage. In most cases, differences between Mal?d?1 of apple fruits stored in the cold chamber and under CA conditions were significant, but inconsistent. In apple cv. ??Elise??, fruits stored in the cold chamber contained more Mal?d?1 compared with those stored in CA, whereas the situation reversed in other varieties like cv. ??Boskoop??. The greatest Mal?d?1 content was measured in fruits of cvs. ??Golden Delicious?? and ??Gala??, whereas the smallest Mal?d?1 level was in cvs. ??Elise?? and ??Pinova?? over the whole storage time. Overall, this experiment showed the complexity of the relationship between the Mal?d?1 content, allergenicity of apple fruits, different cultivars, storage conditions and storage time. Persons allergic to apple fruits should consume the fruits as fresh as possible or only after a limited storage time. Furthermore, they should prefer apple varieties with a low content of allergenic proteins, such as cv. ??Elise?? or cv. ??Boskoop?? as a cultivar known for its large polyphenol content.     

Scrotal circumference,bodyweight and semen characteristics in growing dairy-breed natural-service bulls in Tasmania,Australia

Aims: To provide herd managers with a set of decision rules allowing them to predict the likelihood that a juvenile bull is ready for Bull Breeding Soundness Evaluation (BBSE), or breeding, if bodyweight and scrotal circumference are known.

Methods: This was a longitudinal study following two groups of young pasture-fed Holstein and Jersey bulls from northwest Tasmania, Australia. Individual scrotal circumference, bodyweight and semen characteristics were recorded at 6–8 weekly intervals, from 6–18 months of age. Classification and regression tree analyses were used to predict the probability that a bull had ≥70% normal sperm morphology based on scrotal circumference and bodyweight measurements.

Results: Overall 1,661 scrotal circumference and bodyweight measurements were obtained, and 518 semen samples from 356 bulls were assessed for sperm morphology, from 16 examination sessions that took place between 29 May 2015 and 17 August 2016. Classification and regression tree analyses generated a decision tree for Holstein bulls with four node endpoints, and for Jersey bulls with three node endpoints. Diagnostic test performance showed that for Holstein bulls, using the node endpoints of scrotal circumference ≥27?cm and bodyweight ≥349?kg, 98% had ≥70% normal sperm (positive likelihood ratio 10.4; 95% CI?=?2.7–41), and using the node endpoints of scrotal circumference ≥27?cm and bodyweight between 282–349?kg, 89% had ≥70% normal sperm (positive likelihood ratio 1.6; 95% CI?=?0.9–2.6). For Jersey bulls, using the node endpoints of bodyweight ≥259?kg and scrotal circumference ≥29?cm, 88% had ≥70% normal sperm (positive likelihood ratio 3.4; 95% CI?=?1.6–7.0).

Conclusions: This study provides a set of relatively simple decision rules based on bodyweight and scrotal circumference measurements that allows herd managers to assess the likelihood that juvenile bulls are ready for BBSE or breeding.

Abbreviations: BBSE: Bull breeding soundness evaluation; BRT: Boosted regression tree     

Cross‐sectional observational survey of serum biochemistry values in a population of 69 adult female alpacas (Vicugna pacos) in South Australia

Blood samples were collected from 69 ‘healthy’ female alpacas aged ≥12 months from 11 properties in South Australia. The 10–90 percentile ranges of the 16/19 analytes measured in this sample population were within the published ranges of four healthy alpaca populations from other geographic locations. Marginal exceptions were glutamate dehydrogenase and bicarbonate. Potassium was notably elevated, probably because of haemolysis of some samples. The sample size was insufficient to provide the appropriate statistical power to define diagnostic references ranges according to international standards. The health status of the sample population of alpacas was presumptive based on a physical examination.     

Participation of phosphofructokinase,malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa

The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well‐known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/10¹⁰ spermatozoa, the activity of NAD‐ and NADP‐dependent IDH was 0.111 ± 0.005 U/10¹⁰ and 2.22 ± 0.14 U/10¹⁰ spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/10¹⁰ spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.