Comparison of the omp I gene of Chlamydia psittaci between isolates in Victorian koalas and other animal species

Objective The objective of this study is to compare the strain of chlamydia causing genital infection in koalas from Victoria with isolates from other animal species.
Design Polymerase chain reaction and restriction enzyme analysis has been used to compare various Chlamydia psittaci isolates from a range of animals and disease syndromes. The isolates used in this study include isolates from three birds, three from aborted sheep, one from polyarthritis, one from bovine abortion, one from feline pneumonitis, three porcine isolates from faeces, polyarthritis and abortion, and three urogenital isolates from Victorian koalas.
Procedure Two polymerase chain reactions were performed, each amplifying a different region of the omp I gene. The first polymerase chain reaction amplified a 144 bp segment of the gene which was then digested with the restriction enzyme Eco R I. The second polymerase chain reaction amplified a larger 1070 bp region of the omp I gene which was digested with two restriction enzymes Alu I and Nde II.
Results and conclusions The results obtained have confirmed that variation in DNA sequence of various animal chlamydia isolates does occur. They have also shown that it is possible to classify isolates, based on their restriction enzyme profiles, into distinct groups.     

In vitro Capacitation and Acrosome Reaction of Dog Spermatozoa can be Feasibly Attained in a Defined Medium Without Glucose

Incubation of dog spermatozoa in a medium without glucose and in the presence of lactate and pyruvate (l-CCM) for 4 h at 38.5 degrees C in a 5% CO(2) atmosphere induced in vitro capacitation of these cells. This was verified after the combined specific capacitation-like changes in percentages of viability and altered acrosomes, motility characteristics, sperm location of reactivity against Pisum sativum, Arachis hypogaea and Helix pomatia lectins and the tyrosine phosphorylation pattern. Furthermore, a feasible acrosome reaction (AR) was induced when spermatozoa incubated in l-CCM for 4 h were further co-incubated for 1 h with canine oocytes. This was demonstrated by AR-like changes in percentages of viability, altered acrosomes, motility characteristics and sperm location of reactivity against P. sativum, A. hypogaea and H. pomatia lectins. All these results clearly indicate that in vitro capacitation, and subsequent AR, can be feasibly achieved without the presence of sugars. This ability can be related to the specific characteristics of energy-metabolism regulation reported in dog spermatozoa.     

磷酸泰乐菌素颗粒剂生物效价测定方法的建立及验证

建立了磷酸泰乐菌素颗粒剂的生物效价测定方法，并对新建立的测定方法进行了验证，验证内容包括：方法对照试验、精密度试验、线性及线性范围试验、回收率试验。验证结果证明新方法能准确测定磷酸泰乐菌素颗粒剂的效价单位。     

Influence of Staining Method on the Values of Avian Sperm Head Morphometric Variables

Computer‐assisted systems for the assessment of sperm morphometry (ASMA systems) have been used successfully with several mammalian species. Unfortunately, they have so far been of little use for assessing bird semen, a consequence of the filiform shape of avian spermatozoa. This study compares two staining techniques (Hemacolor^® and aniline blue staining) for the morphometric analysis of rooster and red‐legged partridge spermatozoa as part of a computer‐assisted light microscopy method. For both species, Hemacolor^® staining provided a significantly higher percentage of measurable cells (93.7 ± 11.7% in roosters and 71.9 ± 15.3% in red‐legged partridges). Hemacolor^® also showed greater repeatability (lower coefficients of variation) for length and area in roosters' sperm and for width in the case of red‐legged partridge's sperm. In the roosters, the Hemacolor^® technique returned significantly (p < 0.05) larger sperm head width and area values than did the aniline blue technique, while the latter resulted in greater sperm head length values (p < 0.05). In the red‐legged partridge, no differences were seen in the results for sperm head width and area provided by the two techniques, but aniline blue staining was associated with longer length measurements. In conclusion, the morphometric values recorded differed depending on the staining method and species. However, the Hemacolor^® technique might be deemed the more appropriate for computerized sperm assessment systems as it provides larger percentages of measureable cells and shows greater repeatability.     

Effect of the Interaction Between Cryoprotectant Concentration and Cryopreservation Method on Frozen/Thawed Chicken Sperm Variables

This work examines the effect of the interaction between different concentrations of two cryoprotectants – glycerol (GLY) and dimethylacetamide (DMA) – and two methods of cryopreservation – pellets produced by plunging into liquid nitrogen and gradual in‐straw freezing – on frozen/thawed chicken sperm variables. Sperm was cryopreserved using: (i) 6% DMA, following the in‐straw and the pellet methods (ii) 11% GLY, following the in‐straw and the pellet methods; and (iii) 8% GLY in the in‐straw method and 3% DMA in the pellet method (i.e. reduced cryoprotectant concentrations). When 6% DMA was used as the cryoprotectant, no differences were seen between the in‐straw and pellet methods in terms of frozen/thawed sperm variables or fertility (10.8% and 12.8%, respectively). The viability and motility variables of the frozen/thawed sperm produced using the in‐straw method with 11% GLY were higher (p < 0.05) than those recorded for the sperm preserved using the same cryoprotectant and concentration in the pellet method. However, fertility was extremely low in both groups (2.1% and 4.2% for the in‐straw and pellet methods, respectively). Finally, the use of 8% GLY in the in‐straw method returned higher sperm viability, intact acrosome and motility values than the use of 3% DMA in the pellet method (p < 0.01). No differences were seen, however, in the fertility results obtained (28.8% and 25.0%, respectively). These results suggest that cryoprotectant concentrations can be reduced and still provide acceptable fertility rates.     

A Pilot Study to Compare Oxidative Status between Organically and Conventionally Managed Dairy Cattle During the Transition Period

The aim of this study was to assess the redox balance of organically managed dairy cattle (OMC; n = 40) during the transition period and to compare this with conventionally managed cattle (CMC; n = 22). Serum samples of dairy cows from two organic and one conventional farm were taken. Markers of oxidants production [reactive oxygen species] and total serum antioxidant capacity were measured in four different production stages: (i) far‐off dry (2 to 1 months before calving; 44 samples in CMC and 48 in OMC); (ii) close‐up dry (1 month until 3 days before calving; 44 CMC; 54 OMC); (iii) fresh (3 days to +1 month after calving; 44 CMC; 49 OMC); and (iv) peak of lactation (+1 to +3 months; 71 CMC; 78 OMC). Values were compared between production stages and against a metabolic baseline status (4th–5th month of pregnancy; 40 CMC; 30 OMC). Our results indicated that throughout the periparturient period, OMC had lower concentrations of reactive oxygen species, but also a lower antioxidant capacity than CMC. Indeed, when the two components of the redox balance were assessed together through the Oxidative Stress index, the values of this parameter were higher for OMC than for CMC, thereby implying a higher risk of oxidative stress. Therefore, further larger studies are needed to confirm the current observations, as organically reared animals might be exposed to a lack of antioxidants supply.     

Effect of different mini‐volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer‐assisted semen analyzer

Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 10⁶ sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm^®. A single layer of 40% PureSperm^® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm^® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm^® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm^® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm^® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.     

Clearance of para-aminohippuric acid in wethers consuming locoweed

AIM: To validate the use of para-aminohippuric acid (PAH) as a marker for measuring blood flow in wethers consuming a mixed diet of locoweed and blue grama hay.

METHODS: Fourteen sheep, stratified by bodyweight (BW), were assigned to one of three treatments: 0.8 mg swainsonine (SW)/kg BW (HI), 0.2 mg SW/kg BW (LO), and no SW (Control). Sheep were fed various ratios of locoweed and blue grama hay to deliver SW treatments, for 28 days prior to infusion of PAH. Concentrations of SW and activities of alkaline phosphatase (Alk-P) and aspartate aminotransferase (AST) in serum were measured to confirm exposure to SW and subclinical intoxication. A single 20-ml injection of 5% PAH was delivered into the jugular vein after subclinical intoxication had been achieved. Blood samples were collected and serum analysed for PAH immediately prior to injection, then every 5 min from 5–30 min, and every 10 min from 30–60 min, following injection of PAH.

RESULTS: Effective delivery of SW was evident from the greater concentrations of SW measured in the serum of HI compared with LO animals (p<0.05). No significant differences were detected in the rate of elimination (range 0.097–0.108 L/min), elimination half-life (range 6.62–7.24 min), apparent volume of distribution for the central compartment (range 7.14–9.72 L), and clearance (range 0.73–0.92 L/min) of PAH, between treatments.

CONCLUSIONS: Subclinical intoxication with SW did not affect the pharmacokinetics of PAH. Thus, use of downstream dilution of PAH is a valid method to determine the rate of blood flow in nutrient flux experiments that involve consumption of locoweed.     

Improvement of 2D‐PAGE Resolution of Human,Porcine and Equine Follicular Fluid by Means of Hexapeptide Ligand Library

The major challenge of follicular fluid proteomic analysis is the presence of high‐abundance proteins that originate from plasma. These proteins can prevent the detection of lower abundant ones, produced locally by follicle cells and that may have important roles in follicular activity. In this study, the novel technology called hexapeptide ligand library was evaluated to enrich the low‐abundance proteins in follicular fluid of human (HFF), porcine (PFF) and equine (EFF) prior 2D‐PAGE. Our results showed that the new strategy enabled detection of many new protein spots, increased resolution and highly improved the intensity of low‐abundance proteins by 2D‐PAGE.