Serum testosterone levels in young normal horses

Serum testosterone levels were measured in normal young male horses (29 to 34 weeks old). No differences were found between gelded and intact males. The values for all the horses were low. On the basis of their testosterone levels, all the horses were prepubertal.     

Disposable plastic rodent feeders for use in an automated filling system

Rodent feeder devices are often made of reusable, sanitizable materials such as glass and stainless steel. As part of an initiative at the authors' facility to automate the feeder filling process, disposable plastic feeding cups were proposed as a preferable alternative to standard feeders for use in a filling machine. The authors tested and validated designs for rodent meal-type feeder assemblies that incorporated plastic cups. Assemblies included stainless steel shields that prevented rats and mice from chewing on the plastic cups while allowing them full access to food. The use of disposable feeder cups that are filled and sealed automatically reduces labor associated with sanitation, enables staff to prepare feeders for several weeks in advance and limits personnel contact with feed that may contain harmful chemicals.     

Mitochondrial DNA sequence diversity in extant Irish horse populations and in ancient horses

Equine mitochondrial DNA sequence variation was investigated in three indigenous Irish horse populations (Irish Draught Horse, Kerry Bog Pony and Connemara Pony) and, for context, in 69 other horse populations. There was no evidence of Irish Draught Horse or Connemara Pony sequence clustering, although the majority of Irish Draught Horse sequences (47%) were assigned to haplogroup D. Conversely, 31% of the Kerry Bog Pony sequences were assigned to the rare haplogroup E. In addition to the extant population analyses, ancient DNA sequences were generated from three out of four Irish archaeological specimens, all of which were assigned to haplogroup A.     

Learning performances in young horses using two different learning tests

To achieve optimal performance in equine sports as well as in leisure not only the physical abilities of the horse should be considered, but also the horse’s personality. Besides temperamental aspects, like emotionality, or the horse’s reactivity towards humans in handling situations, the learning ability of the horse is another relevant personality trait. To study whether differences in learning performance are consistent over time and whether individual learning performance differs between learning tests or is affected by emotionality, 39 young horses (Dutch Warmblood) were tested repeatedly in two learning tests. An aversive stimulus (AS) was used in one learning test (the avoidance learning test) and a reward was used in the other learning test (the reward learning test). During both learning tests behaviour as well as heart rate were measured. Each test was executed four times, twice when horses were 1 year of age, and twice when they were 2 years of age. Half of the horses received additional physical training from 6 months onwards. In both tests horses could be classified as either performers, i.e. completing the daily session, or as non-performers, i.e. returning to the home environment without having completed the daily session. There were some indications that emotionality might have caused non-performing behaviour, but these indications are not convincing enough to exclude other causes. Furthermore, there seem to be no simple relationships between measures of heart rate, behavioural responses putatively related to emotionality and learning performance. Horses revealed consistent individual learning performances within years in both tests, and in the avoidance learning test also between years. There was no significant correlation between learning performances in the avoidance learning test and the learning performances in the reward learning test. It is concluded that individual learning abilities are consistent over a short time interval for an avoidance learning test and a reward learning test and over a longer time for the avoidance learning test. Furthermore, results indicate that some horses perform better when they have to learn to avoid an aversive stimulus while others perform better when they are rewarded after a correct response. It is suggested that these differences may be relevant to design optimal individual training programmes and methods.     

Boots on horses: limb protection or hyperflexion training aids in the showjumping horse

Showjumping riders regularly employ various schooling strategies to control the horse's jump stride kinematics (JSK). Strategies include plyometric training regimes with fences of different heights and widths set at specific distances. Gymnastic grids teach the horse to jump cleanly. Rapping, once used almost routinely, is no longer in vogue. However, the use of performance enhancing (PE) boots on the distal hind limbs to alter equine JSK has become popular. There are two broad categories of PE boots: weighted and pressure. Some riders use so-called weighted boots on the horses' hind limbs during training and in competition to improve the jump stride. The application of so-called pressure boots may be little more than an adaptation of this technique. It appears that the PE boots induce hyperflexion of the hind limbs and incline the horse to jump fences cleanly. In the absence of scientific appraisal, it is unclear if such boots are acceptable and innovative training aids within equitation.     

Attempts to use the HPRT-assay as an automated short-term monitor for an acute exposure to mutagens

Attempts have been made to use the hypoxanthine-guanine-phospho-ribosyl-transferase-assay as a method for automated screening of agent-induced phenotypic variants of human peripheral lymphocytes reflecting 6-thioguanine resistance and assumed to indicate genotoxic action. Different protocols of the hypoxanthine-guanine-phospho-ribosyl-transferase-system were used in this study in order to investigate whether the system can be a candidate for a short-term test for a rapid and reliable identification of biological systems exposed to agents. The current protocols were based on: 1) fluoresceinated monoclonal antibodies against bromodeoxyuridine-DNA for labelling of 6-thioguanine-resistant human lymphocytes and direct flow-cytometric enumeration of bromodeoxyuridine-positive events and: 2) indirect flow-cytometric enrichment of 6-thioguanine-resistant cells labelled with ³H-thymidine followed by autoradiographic enumeration of positive events. Both the direct and the indirect enumeration method yielded similar results down to the range 10^–4 with respect to frequency of variants. For the less time-consuming direct enumeration method the resolution was limited due to non-specific binding of the antibody and false positives. It was, nevertheless, sufficient to score variants induced in vitro with the mutagens EMS, MMC and TT in the same range as e.g. that of cancer patients during and after chemotherapy or radiotherapy, or that of psoriasis patients during and after PUVA (8-methoxypsoralen and long range UV light)-therapy. We conclude that the direct enumeration protocol can be used for a rapid screening of so called outliers, but a more sensitive test, such as the more time-consuming enrichment protocol based on autoradiography, must be used in order to score variants in the range 10^–5–10^–6Abbreviations ³H-TdR Tritiated thymidine - BrdU 5-2-bromodeoxyuridine - DNA Deoxyribonucleic acid - EMS Ethyl-methanesulphonate - FBS Fetal bovine serum - FITC Fluorescein Isothiocyanate - G₀ Cell cycle stage - G₁ Cell cycle stage - G₂ Cell cycle stage - HPRT Hypoxanthine Guanine Phospho Ribosyl Transferase - LI Labelling index - MMC Mitomycin C - PBL Peripheral blood lymphocytes - PBS Phosphate buffered saline - PHA Phytohemagglutinin - PI Propidium iodide - PUVA 8-Methoxypsoralen and long range UV-light therapy - RNA Ribonucleic acid - RPMI RPMI-1640 culturing medium - S_e Early S-stage in the cell-cycle - S_l Late S-stage in the cell-cycle - SCE Sister chromatid exchange - TG 6-Thioguanine - TT Thiotepa - UV Ultraviolet light - V_f Variant frequency     

The use of an automated assay to assess phage survival after a biocidal treatment

The coliphage K_1-5 has been used in an automated assay to monitor the viricidal activity of various disinfectants. This indirect assay based on the spectrophotometric reading of the lysis of the host cell ( Escherichia coli D837) produced encouraging results and was faster than the overlay counting method (previously studied) which relies on plaque formation. However, differences in sensitivity towards some disinfectants were observed between the two methods.     

Prevalence of antibodies to Neospora sp. in horses from Alabama and characterisation of an isolate recovered from a naturally infected horse [corrected

An IFAT was used to determine the prevalence of Neospora-specific IgG antibodies in serum from Alabama horses. Serum samples (n = 536) were from asymptomatic horses routinely submitted for equine infectious anaemia virus infection testing. We also subjected a 13-year-old horse with CNS disease to necropsy examination for isolation and in vitro cultivation of protozoal organisms. In antemortem tests, this horse was positive for antibodies to Neospora sp. in the IFAT and western immunoblot. Results of the prevalence survey indicated that IgG antibodies to Neospora were present in 62 (11.5%) of the 536 serum samples. Endpoint titres for the positive samples were 1:50 (35/6.5%), 1:100 (19/3.5%), 1:200 (7/1.3%) and 1:1600 (1/0.2%). Tachyzoites were first seen in cultured bovine turbinate cells 32 days after inoculation with spinal cord homogenates from the horse with CNS disease. Tachyzoites reacted with known N. caninum-positive serum from horses, cows, dogs and mice, but did not react with murine anti-Toxoplasma gondii or equine anti-Sarcocystis neurona serum. Ultrastructural features of tachyzoites and results of comparison of tachyzoite immunodominant proteins revealed that they were identical to those of N. hughesi, a species described recently from a naturally infected horse. The isolate recovered from the naturally infected horse in the present study (designated NA1) is thought to be an isolate of N. hughesi, although confirmation of this awaits additional molecular characterisation. These results provide some additional evidence that N. hughesi is a valid species and that Neospora infections in horses may occur in widely separated geographic regions of the United States.     

Microsatellite variation in Japanese and Asian horses and their phylogenetic relationship using a European horse outgroup

The genetic relationships of seven Japanese and four mainland-Asian horse populations, as well as two European horse populations, were estimated using data for 20 microsatellite loci. Mongolian horses showed the highest average heterozygosities (0.75-0.77) in all populations. Phylogenetic analysis showed the existence of three distinct clusters supported by high bootstrap values: the European cluster (Anglo-Arab and thoroughbreds), the Hokkaido-Kiso cluster, and the Mongolian cluster. The relationships of these clusters were consistent with their geographical distributions. Basing our assumptions on the phylogenetic tree and the genetic variation of horse populations, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The genetic relationship of Japanese horses corresponded to their geographical distribution. Microsatellite polymorphism data were shown to be useful for estimating the genetic relationships between Japanese horses and Asian horses.     

Linkage of hyperkalaemic periodic paralysis in Quarter horses to the horse adult skeletal muscle sodium channel gene

A genetic disease observed in certain Quarter horses is hyperkalaemic periodic paralysis (HYPP). This disease causes attacks of paralysis which can be induced by ingestion of potassium. Recent studies have shown that HYPP in humans is due to single base changes within the adult skeletal muscle sodium channel gene. A large Quarter horse pedigree segregating dominant HYPP was studied to determine if mutations of the sodium channel gene are similarly responsible for HYPP in horses. We used cross-species, PCR-mediated, cDNA cloning and sequencing of the horse adult skeletal muscle sodium channel alpha-subunit gene to identify a polymorphism, and then used this polymorphism to see if the horse sodium channel gene was genetically linked to HYPP in horses. The sodium channel gene was indeed found to be tightly linked to HYPP (LOD = 2.7, theta = 0). Our results suggest that HYPP in horses involves the same gene as the clinically similar human disease, and indicates that these are homologous disorders. The future identification of the specific sodium channel mutation causing HYPP in Quarter horses will permit the development of accurate molecular diagnostics of this condition, as has been recently shown for humans.     

The use of immobilized enzyme-membrane sandwich reactors in automated analysis

Glucose oxidase from A. niger (EC 1.1.3.4) was physically entrapped between two dialysis membranes. The resultant enzyme-membrane sandwich reactors were employed in a standard Technicon dialyzer unit at 37°C and were found to perform efficiently in the automated analysis of aqueous glucose standards in the range 2.5–50 mg%. Over a 10-day period with intermittent usage, slight increases in the activities of the reactors were observed. The potential of such reactors in automated analysis is discussed.     

The use of an automated growth analyser to measure recovery times of single heat-injured Salmonella cells

A new approach to the study of recovery times of single heat-injured Salmonella cells isdescribed. It comprises the generation of a standard heat-injured culture, serial dilution of thisculture to near extinction, inoculation of the serial dilutions across many microtitre plates andmeasurement of the subsequent recovery and growth using an automated turbidometric analyser.Lag times for individual cells were estimated from turbidity data using a model that accuratelyextrapolated the growth curve back to the starting inoculum level. Lag times were comparedusing a number of different commercially available pre-enrichment media. The most typical resultwas a very broad distribution of lag times at the single cell inoculum level, with many values inexcess of 20 h. Even at an inoculum level 10-fold higher, lag times for some injured cells wereestimated to be >10 h. More significantly, it was found that some media recovered more injuredcells than others and vice versa. Between the worst and best media there were as many as 3 log₁₀ cycles difference in the number of cells recoverable. No trends were apparent linkingchoice of medium with performance. The implications of these findings, in relation to traditionaland rapid methodology, are discussed.     

Occurrence of an unusual phosphorylated N-acetyllactosamine in horse colostrum

The colostrum of horses (thoroughbreds) was extracted and fractionated to yield Gal(beta1-4)GlcNAcalpha1-phosphate, which has not previously been detected in any mammalian milk or colostrum, as well as Neu5Ac(alpha2-3)Gal(beta1-4)Glc. The structures of these saccharides were established by NMR spectroscopy and MALDI-TOF mass spectrometry.     

Phylogenetic relationships of Cheju horses to other horse breeds as determined by mtDNA D-loop sequence polymorphism

Historical records suggest that horses inhabiting the island of Cheju in Korea are descendants of Mongolian horses introduced in 1276. Other studies, however, suggest that horses may have been present on the island prior to the Mongolian introduction. To determine the origin of the Cheju horses we used a phylogenetic analysis of sequences of the mitochondrial DNA (mtDNA) D-loop region, including tRNA Pro and parts of tRNA thr and tRNA Phe sequences (1102-bp excluding the tandem repeat region). Maximum parsimony and neighbor-joining trees were constructed using sequences determined for seven Cheju, four Mongolian, one Przewalskii and two Chinese Yunnan horses, and published sequences for one Swedish and three Thoroughbred horses. Donkey mtDNA was used as an outgroup. We found that the mtDNA D-loop sequence varies considerably within Mongolian, Cheju and Thoroughbred horse breeds, and that Cheju horses clustered with Mongolian horses as well as with horses from other distantly related breeds. On the basis of these findings we propose that horses on Cheju Island are of mixed origin in their maternal lineage, and that horses may have existed and been traded on the island before the Mongolian introduction.     

Toluene removal in an automated cyclical bioreactor

A control scheme was developed for the automation of toluene removal in a cyclical bioreactor. Toluene was added to the self-cycling fermentor by diffusion across a silicone membrane. Transient dissolved oxygen, carbon dioxide evolution, and oxidation-reduction potential (ORP) were screened as potential control variables. Through experimentation, ORP was deemed most effective. Control algorithms based on real-time estimates of the first and second derivatives of the ORP signal were tested. Although both approaches resulted in stable operation of the reactor, average toluene removal efficiencies of 95% were realized when control was based on the second derivative. This was significantly higher than the 77% efficiencies obtained when the control scheme centered on the first derivative of the transient ORP signal. The system developed was self-regulating, ensuring that a high toluene removal rate, on the order of 1.1 g h(-1), was maintained from cycle to cycle.     

Mechanism of substrate sensing and signal transmission within an ABC transporter: use of a Trojan horse strategy

By translocating proteasomal degradation products into the endoplasmic reticulum for loading of major histocompatibility complex I molecules, the ABC transporter TAP plays a focal role in the adaptive immunity against infected or malignantly transformed cells. A key question regarding the transport mechanism is how the quality of the incoming peptide is detected and how this information is transmitted to the ATPase domains. To identify residues involved in this process, we evolved a Trojan horse strategy in which a small artificial protease is inserted into antigenic epitopes. After binding, the TAP backbone in contact is cleaved, allowing the peptide sensor site to be mapped by mass spectrometry. Within this sensor site, we identified residues that are essential for tight coupling of peptide binding and transport. This sensor and transmission interface is restructured during the ATP hydrolysis cycle, emphasizing its important function in the cross-talk between the transmembrane and the nucleotide-binding domains. This allocrite sensor may be similarly positioned in other members of the ABC exporter family.