Glycerol,Methyl‐Formamide and Dimethyl‐Formamide in Canine Semen Cryopreservation

The aim of the present work was to compare the efficiency of methyl‐formamide (MF), dimethyl‐formamide (DF) and glycerol (GL) as cryoprotectants in canine semen cryopreservation. For the experiment, pooled semen was submitted to one of the three cryoprotectants, with a final concentration of 3% in egg yolk–TRIS extender. Semen was subjectively evaluated for total and progressive motility, vigour and morphology. Sperm membrane functional integrity was assessed by hypo‐osmotic swelling test (HOST), and longevity was assessed using the thermoresistance test (TRT). Fresh semen showed normal physical and morphological characteristics. After thawing, differences were observed between semen frozen using GL and DF, regarding total and progressive motility and vigour (p < 0.05), but not between MF and GL or MF and DF. Means for total motility, progressive motility, vigour and morphologically normal spermatozoa were, respectively, 69.0 ± 5.4%, 61.0 ± 7.4%, 2.9 ± 0.5 and 57.1 ± 5.0% for GL; 59.0 ± 8.9%, 50.0 ± 10.0%, 2.5 ± 0.7 and 66.9 ± 7.7% for MF; and 44.0 ± 21.0%, 37.0 ± 19.8%, 2.1 ± 0.6 and 61.1 ± 5.5% for DF. On HOST, GL was superior (p < 0.05) to MF and DF (57.8 ± 12.4%, 35.8 ± 18.4% and 34.4 ± 9.4%, respectively). During the TRT, both GL and MF were superior to DF, with no differences between GL and MF. In conclusion, the use of MF as cryoprotectant showed results similar to GL, and can be considered as an alternative in canine semen cryopreservation. Further studies testing different concentrations of MF may improve its effects on cryopreservation of canine semen.     

Efficacy of intramuscular polysulfated glycosaminoglycan in a controlled study of equine carpitis

Verde, C., Ferrante, M., Simpson, M. I., Babusci, M., Broglia, G., Landoni I, M. F. Efficacy of intramuscular polysulfated glycosaminoglycan in a controlled study of equine carpitis. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365‐2885.2009.01154.x. Twelve healthy horses were subject to the monoioidoacetate (MIA) carpitis model, which was allowed to develop for 7 days. The horses were then randomly divided into two groups. Group A (control) received an intramuscular injection of normal saline every 4 days for a total of seven injections while group B received 500 mg of a PSGAG (SYNTEX CSY36) intramuscularly every 4 days for seven treatments. Efficacy of the PSGAG was evaluated by three clinical outcomes: lameness score, carpal circumference and maximum carpal flexion. Clinical outcomes were measured on days ‐8 (previous to carpitis induction), 0 (previous to drug treatment), 7, 14, 21, 28 and 35. Areas under the curve clinical outcome as function of time were built and used as variables for the statistical analysis. There was less joint circumference enlargement and lameness and greater carpal flexion in PSGAG‐treated horses compared with that in controls. The studied compound has demonstrated to be efficacious on the treatment of a chemically induced carpitis in horses.     

Proliferation and Apoptosis in Aged and Photoregressed Mammalian Seminiferous Epithelium,with Particular Attention to Rodents and Humans

Imbalances in the proliferation and apoptosis processes are involved in numerous epithelial alterations. In the seminiferous epithelium, normal spermatogenesis is regulated by spermatogonia proliferation and germ cell apoptosis, and both processes are involved in diverse pathological alterations of the seminiferous epithelium. Other physiological phenomena including aging and short photoperiod, in which apoptosis and proliferation seem to play important roles, cause testicular changes. Aging is accompanied by diminished proliferation and increased apoptosis, the latter occurring in specific states of the seminiferous cycle and considered the cause of epithelium involution. However, there is no clear evidence concerning whether proliferation decreases in the spermatogonia themselves or is due to an alteration in the cell microenvironment that surrounds them. As regards the factors that regulate the process, the data are scant, but it is considered that the diminution of c‐kit expression in the spermatagonia, together with the diminution in antiapoptotic factors (Bcl‐x_L)) of the intrinsic molecular pathway of apoptosis play a part in epithelial regression. A short photoperiod, especially in rodents, produces a gradual involution of the seminiferous epithelium, which is related with increased apoptosis during the regression phase and a diminution of apoptosis during recrudescence. Proliferative activity varies, especially during the total regression phase, when it usually increases in the undifferentiated spermatogonia. In other species showing seasonal reproduction, however, decreased proliferation is considered the main factor in the regression of the seminiferous epithelium. Little is known about how both phenomena are regulated, although data in rodents suggest that both the intrinsic and extrinsic pathways of apoptosis contribute to the increase in this process. In conclusion, regression of the seminiferous epithelium in physiological situations, as in many pathological situations, is a result of alterations in equilibrium between the proliferation and apoptosis of germinal cell types. However, both physiological phenomena showed important differences as regard proliferation/apoptosis and their regulation pathways, probably as a result of their irreversible or reversible character.     

The relationship of training milestones with racing success in a population of Standardbred horses in New Zealand

AIM: To gather information on the repeatability of a faecal nematode egg count (FEC) reduction (FECR) test (FECRT), evaluating both different methods of calculating efficacy and variations within a method, in order to supply veterinarians and other advisors with sufficient information to apply some level of confidence around a diagnosis of anthelmintic resistance based on FECRT results.

METHODS: Two commercial sheep farms were selected on the basis of having previously recorded FECR <95% after treatment with ivermectin (Farm 1) or albendazole (Farm 2). On each farm at least 250 lambs, managed as a single mob, were individually ear-tagged and sampled for FEC. The resulting counts were used, 3—4 days later, to sort the lambs into 24 groups of 10. First, the animals were split into three groups of 80, having high, medium or low FEC. Second, within each of these groups the 80 animals were further divided into four replicate mobs of 20 (each with the same mean count). Third, each of these replicates was further split into two groups of 10: those that would be drenched and those that would remain as untreated controls. All animals were again faecal-sampled and those in the drenched groups were dosed, using a syringe, to their individual liveweight, with ivermectin (Farm 1) or albendazole (Farm 2). Ten days after treatment all animals were individually faecal- sampled again. FEC and larval cultures were undertaken for all 24 groups from both pre- and post-treatment samples. Ef- ficacy (FECR) of the undifferentiated FECRT was calculated using three different equations, and efficacy by genus was also calculated.

RESULTS: Calculated efficacies differed between equations, and the equation which did not utilise an untreated control yielded significantly lower efficacy estimates on both farms. Faecal cultures varied considerably in the proportions of parasite genera recovered. In general, this did not differ between FEC groups, except on Farm 1 where Haemonchus spp were more common and Cooperia spp less common in high-FEC samples. Estimated efficacies against individual genera varied considerably or very little, depending on the level of resistance. On both farms, differing proportions of tests against some genera passed or failed FECRTs based on a threshold pass mark of ≥95% FECR.

CONCLUSION: There was considerable variability in the outcomes of FECRTs and in larval culture results. Caution is warranted in interpreting the results of FECRTs when efficacy values fall into the 90—95% range. Further, the possibility of a test returning a false-negative result is raised, indicating that even an efficacy estimated ≥95% may not guarantee the absence of resistant parasites.     

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene.

METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay.

RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 10⁴ cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%.

CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.     

Dynamics of the free-living stages of sheep intestinal parasites on pasture in the North Island of New Zealand. 1. Patterns of seasonal development

Abstract

AIM: To describe the seasonal pattern of development of third-stage infective larvae (L3) from eggs of Teladorsagia (=Ostertagia) circumcincta, Trichostrongylus colubriformis and Haemonchus contortus on pasture in the North Island of New Zealand.

METHODS: Sheep faeces containing known numbers of eggs of all three nematode species were deposited on, or buried in, pasture plots at three sites, viz coastal Manawatu, Upper Hutt Valley, and East Cape hill country. Development was measured by recovering L3 from faeces, herbage and soil 28–31 days after deposition on 13–18 occasions, between January 2005 and July 2006. Analysis of the number of larvae recovered used a mixed model including number of eggs deposited, weight of faeces recovered (an assumed indicator of earthworm activity), site, contamination date, and position of deposited faeces, i.e. on the surface or buried.

RESULTS: There was a significant effect of contamination date on development of all three species, with maximum numbers ofL3 developing between late spring (November) and early autumn (March), and minimum numbers in June and July. There were large differences between species, with H. contortus exhibiting a long period (April to October) where development was close to zero, whereas T. circumcincta developed to some extent all year round. Development of T. colubriformis was intermediate between the other two species.

Burying faeces containing nematode eggs increased the number of L3 recovered compared with surface deposition (p≤0.001), although there were a small number of exceptions involving only T. colubriformis. The weight of faeces recovered at harvest, which was assumed to be an indication of earthworm activity, was correlated with the number of L3 recovered for all species (p<0.001). In a separate analysis, earthworms were assumed tohave been active if <5 g faeces remained at harvest. Where this occurred, the number of L3 of T. colubriformis and T.circumcincta recovered was reduced by 56% and 58%, respectively (p<0.001).

CONCLUSIONS: A marked seasonal pattern of development was observed for all three species, with the most larvae developing in spring-early autumn and the least in winter. This seasonal pattern was most pronounced in H. contortus and least obvious in T. circumcincta. Burying faeces containing eggs generally resulted in more L3 being recovered, whilst the apparent activity of earthworms resulted in fewer larvae being recovered.     

Development and spatial distribution of the free-living stages of Teladorsagia circumcincta and Trichostrongylus colubriformis on pasture: A pilot study

Abstract

AIMS: To measure the development of Teladorsagia (=Ostertagia) circumcincta and Trichostrongylus colubriformis eggs to third-stage infective larvae (L3) at different times of the year. Also, to measure the spatial distribution of L3 across herbage, soil and faeces, in order to assess whether spatial issues could be important in larval dynamics on pasture.

METHODS: Field plots were contaminated with sheep faeces containing approximately 20,000 eggs of each of T. circumcincta and T. colubriformis on five separate occasions, viz 01 December 1996 (summer), 18 March 1998 (autumn), 17 June 1998 (winter), 15 October 1998 (spring), and 23 July 1999 (winter). Replicate plots (n=10) were harvested at intervals for up to 12 months after deposition of faeces, and the number and distribution of L3 were measured. Larvae were sampled from faeces (where these remained), herbage, and three soil zones to a depth of 145 mm.

RESULTS: There were large differences between contamination dates in the percentage of eggs that developed to L3. For both species the highest percentage development was for eggs deposited in December (7.8% and 25.9% for T. circumcincta and T. colubriformis, respectively) and the lowest for June (0.4% and 0.03% T. circumcincta and T. colubriformis, respectively). Development in winter was often delayed, and this was always associated with a low yield of larvae, probably due to compounding mortalities associated with long periods of exposure to low temperatures.

The relative distribution of L3 present on herbage, in faeces or in the soil varied between sampling times. However, overall the most L3 were recovered from soil (74% and 66% for T.circumcincta and T. colubriformis, respectively, averaged over all samples), and the lowest recoveries were from the herbage.

CONCLUSIONS: Although the data are limited, the results indicated that the highest percentage of eggs developed to infective larvae in summer and only minimal development occurred in winter. The data do not support the view that substantial contamination of pastures with sheep parasites occurs over winter. Large numbers of larvae were recovered from soil, which indicates that, assuming they can subsequently migrate onto herbage, soil is a potentially important reservoir ofinfective larvae in New Zealand. Therefore, the spatial distribution of L3 on pasture may affect both the dynamics and transmission of parasite populations. Further work on both these issues is warranted.