Welfare assessment in dairy cows using hair cortisol as a part of monitoring protocols.

Welfare of dairy cows can be assessed using welfare assessment protocols consisting of resource, management and animal-based measures. Welfare Quality® Assessment Protocol is one of the best-known protocols, which depends almost entirely on animal-based measures. To gain more objective and rapid welfare assessment, new techniques have been developed to measure welfare of animals, such as hair cortisol concentration. As cortisol is released in response to stress, it has long been used as a biomarker of stress in animals. While the precise mechanism of cortisol incorporation into hair is unknown, hair cortisol concentration seems to be a marker of long-term systemic cortisol concentration. Hair cortisol is, therefore, a potential marker of chronic stress and is not likely to be affected by acute stress. Studies on cattle show connections between hair cortisol concentration and factors such as pregnancy, parity, diseases, ectoparasites, body condition score, environmental changes, stocking density and milk yield. Hair cortisol concentration appears to be affected by time of sampling, cow age and breed, UV radiation, season, body region of sampled hair and hair colour. Its concentration also depends on sampling and analytical methods. Hair cortisol is a promising non-invasive tool to evaluate welfare of dairy cows, however, more research is needed to determine the extent of effects on its concentration and the appropriate method of sampling and analysis. Correlations between Welfare Quality® Assessment Protocol scores and pooled hair cortisol concentrations have not yet been found, and more research is needed with larger sample size, a standardized protocol of hair sampling, processing and analysis. With proper attention to detail, hair cortisol levels in pooled hair samples might come to be used as a reliable indicator of dairy animal welfare.

Simplifying sampling for African swine fever surveillance: Assessment of antibody and pathogen detection from blood swabs.

African swine fever (ASF) is a notifiable disease with serious socio-economic consequences that has been present in wild boar in the Baltic States and Poland since 2014. An introduction of ASF is usually accompanied by increased mortality, making fallen wild boar and hunted animals with signs of disease the main target for early warning and passive surveillance. It is difficult, however, to encourage hunters and foresters to report and take samples from these cases. A pragmatic and easy sampling approach with quick-drying swabs could facilitate this. In this study, we further evaluated the use of dry blood swabs for the detection of ASFV antibody and genome with samples from animal trials and diagnostic submissions (blood, bone and organs) from Estonia. Compared to serum samples, dried blood swabs yielded 93.1% (95% confidence interval: [83.3, 98.1]) sensitivity and 100% [95.9, 100.0] specificity in a commercial ASFV antibody ELISA. Similarly, the swabs gave a sensitivity of 98.9% [93.4, 100.0] and a specificity of 98.1% [90.1, 100.0] for genome detection by a standard ASFV p72 qPCR when compared to EDTA blood. The same swabs were tested in a VP72-antibody lateral flow device, with a sensitivity of 94.7% [85.4, 98.9] and specificity of 96.1% [89.0, 99.2] compared to the serum ELISA. When GenoTube samples tested in ELISA and LFD were compared, the sensitivity was 96.3% [87.3, 99.5] and the specificity was 93.8% [86.0, 97.9]. This study demonstrates reliable detection of ASFV antibody and genome from swabs. A field test of the swabs with decomposed wild boar carcasses in an endemic area in Estonia also gave promising results. Thus, this technique is a practical approach for surveillance of ASF in both free and endemic areas.

A Low-Cost Method of Skin Swabbing for the Collection of DNA Samples from Small Laboratory Fish.

Fin clipping of live fish under anesthesia is widely used to collect samples for DNA extraction. An alternative, potentially less invasive, approach involves obtaining samples by swabbing the skin of nonanesthetized fish. However, this method has yet to be widely adopted for use in laboratory studies in the biological and biomedical sciences. Here, we compare DNA samples from zebrafish Danio rerio and three-spined sticklebacks Gasterosteus aculeatus collected via fin clipping and skin swabbing techniques, and test a range of DNA extraction methods, including commercially available kits and a lower-cost, in-house method. We verify the method for polymerase chain reaction analysis, and examine the potential risk of cross contamination between individual fish that are netted together. We show that swabbing, which may not require the use of anesthesia or analgesics, offers a reliable alternative to fin clipping. Further work is now required to determine the relative effects of fin clipping and swabbing on the stress responses and subsequent health of fish, and hence the potential of swabbing as a refinement to existing DNA sampling procedures.

Assessment of litter prevalence of Mycoplasma hyopneumoniae in preweaned piglets utilizing an antemortem tracheobronchial mucus collection technique and a real-time polymerase chain reaction assay.

The swine industry currently lacks validated antemortem methods of detecting baseline herd prevalence of Mycoplasma hyopneumoniae. The focus of our study was to evaluate alternative antemortem detection techniques and to determine baseline litter prevalence in preweaned pig populations utilizing the selected technique and a real-time polymerase chain reaction (qPCR) assay. Preliminary data was analyzed on weaned piglets with evidence of respiratory disease (n = 32). Five sample types (antemortem nasal swab, tracheobronchial mucus, postmortem deep airway swab, bronchoalveolar lavage, and lung tissue) were collected from each pig. Individual samples were tested for M. hyopneumoniae using qPCR. Compared to nasal swabs, tracheobronchial mucus demonstrated higher test sensitivity (P < 0.0001). Tracheobronchial mucus was collected from apparently healthy preweaned piglets (n = 1,759; sow farms 1-3) and preweaned piglets exhibiting signs of respiratory disease (n = 32; sow farm 4), ranging in age from 12 to 25 days. Samples from sow farms 1-3 were pooled into 2 groups of 5 per litter (n = 360 pools from 180 litters), and qPCR was utilized to analyze each pool. A qPCR-positive result, threshold cycle <37, from either pool designated the litter positive for M. hyopneumoniae. Two out of 180 litters revealed a positive result (1.1%). Individual qPCR assays were run on the samples collected from sow farm 4. Five out of 30 samples revealed a positive result (16.7%). Tracheobronchial mucus collection in combination with qPCR is a sensitive antemortem sampling technique that can be used to estimate the prevalence of M. hyopneumoniae in preweaned pigs, thus providing insight into the infection dynamics across the entire farrow-to-finish process.

Assessment of two different types of sample for the early detection and isolation of thermophilic Campylobacter in broiler farms.

In order to assess the optimal method for the early detection and isolation of thermophilic Campylobacter in broilers at farm level, two types of samples were compared: caecal contents obtained by necropsy and cloacal swabs transported in charcoal Amies medium. The study was conducted in five batches of broilers from five different farms, where weekly samples (caecal contents and cloacal swabs) from 30 birds were obtained. Samples were plated onto selective agar (modified charcoal cefoperazone desoxycholate agar, mCCDA) for Campylobacter isolation. Four out of five batches were positive for Campylobacter. No marked differences in sensitivity of both sample types were observed. However, a higher percentage of positive birds were detected when cloacal swabs were used. The results show that cloacal swab samples are adequate, and in some cases even better than caecal samples for the early detection of Campylobacter in broiler flocks at farm level. Also, this sample avoids sacrificing birds to test Campylobacter, which not only allows saving time in sample collection, transportation and processing at the laboratory, but also improves bird welfare and cost of sampling.

Evaluation of the impact of refrigeration on next generation sequencing-based assessment of the canine and feline fecal microbiota.

BACKGROUND: Evaluation of factors that might impact microbiota assessment is important to avoid spurious results, particularly in field and multicenter studies where sample collection may occur distant from the laboratory. This study evaluated the impact of refrigeration on next generation sequence-based assessment of the canine and feline fecal microbiota. Fecal samples were collected from seven dogs and ten cats, and analysed at baseline and after 3, 7, 10 and 14 days of storage at 4°C. RESULTS: There were no differences in community membership or population structure between timepoints for either dogs or cats, nor were there any differences in richness, diversity and evenness. There were few differences in relative abundance of phyla or predominant genera, with the only differences being significant increases in Actinobacteria between Days 0-14 (P = 0.0184) and 1-14 (P = 0.0182) for canine samples, and a decrease in Erysipelotrichaceae incertae sedis between Day 0 and Day 7 (median 4.9 vs 2.2%, P = 0.046) in feline samples. CONCLUSIONS: Storage for at least 14 days at 4°C has limited impact on culture-independent assessment of the canine and feline fecal microbiota, although changes in some individual groups may occur.

Comparison of individual and pooled faecal samples in sheep for the assessment of gastrointestinal strongyle infection intensity and anthelmintic drug efficacy using McMaster and Mini-FLOTAC.

A field study was conducted to validate pooled faecal samples in sheep for the assessment of gastrointestinal (GI) strongyle infection intensity (faecal egg count - FEC) and anthelmintic drug efficacy (FEC reduction - FECR). Ten sheep farms located in the Campania region of southern Italy were selected for the study. In each farm, individual faecal samples from 20 adult sheep (when possible) were collected, before (D0) and after (D14) an anthelmintic treatment with albendazole. For each farm and at each time point (D0 and D14) the faecal samples were examined individually and as pools. Specifically, three different pool sizes (5, 10 and 20 individual sheep samples) and three different analytic sensitivities (namely 10 using Mini-FLOTAC; 15 and 50 using the two variants of McMaster - McM15 and McM50) were compared for FEC and FECR using individual and pooled faecal samples. GI strongyle intensity (eggs per gram of faeces - EPG) of pooled samples correlated positively with mean EPG of individual samples, with very high correlation coefficients (ranging from 0.94 to 0.99) across the 3 different pool sizes and analytic sensitivities. Mini-FLOTAC was more sensitive compared to the two variants of McMaster (McM15 and McM50) for the diagnosis of GI strongyles in sheep (100% vs 88.5% vs 75.9%) and resulted in significant higher FEC compared to both McM15 and McM50, with a mean difference in egg counts of approximately 90 EPG (p<0.001). The drug efficacy results showed that FECR was higher than 98% at most farms independently of the pool size and analytic sensitivity. With the exception of two farms, FECR was 100% when calculated for individual animals and across the different pool size and analytic sensitivities. In conclusion, the present study highlighted that pooling ovine faecal samples is a rapid procedure that holds promise as a valid strategy for assessing GI strongyles FEC and FECR in sheep.

A protocol for the management of canine cerebrospinal fluid for the proteomic assessment of putative biomarkers.

Cerebrospinal fluid (CSF) is a potential source for disease-specific biomarkers that may assist in the staging and determining the prognosis of neurodegenerative conditions in animals. However, the validity of such putative biomarkers may be influenced by pre-analytical variables, including the procedures adopted to collect and store the CSF. This study assessed the effect of three handling practices on the stability of a panel of CSF proteins: clusterin (also known as apolipoprotein J), haptoglobin, cystatin C, and transthyretin (TTR). The three handling procedures for canine CSF were mimicked in the laboratory as follows: (1) storage in a refrigerator overnight (4 °C for 18 h); (2) carrying a sample in the pocket of a clinician (37 °C for 4h); and (3) mailing a sample to a remote laboratory for analysis (room temp for 48 h). The impact of these three scenarios on the concentrations of the selected proteins was assessed using Western blotting and compared to an aliquot of CSF that had been kept frozen. The level of clusterin was significantly reduced following 48 h at room temperature (P<0.05), while the concentration of the dimeric form of TTR increased following this handling procedure and also when held at 37 °C for 4h. A reducing agent prevented this increase at 37 °C. In conclusion, exposing CSF samples to various environmental conditions can significantly alter their protein content, a factor that must be considered in studies assessing potential biomarkers in canine CSF.

Assessment of routine procedure effect on breathing parameters in mice by using whole-body plethysmography.

We used whole-body plethysmography to investigate the effect of restraint, ear marking, tail vein and retroorbital blood sampling, and tail clipping on respiration in Balb/c × TCR-HA +/- F1 hybrid mice (F1h). Baseline values of breathing parameters were determined. During the experiment, mice experienced a procedure and then plethysmographic recordings were obtained immediately and at 4, 24, and 48 h afterward. Baseline breathing parameters showed significant differences between sexes. Restraint affected minute volume differently than did handling in male mice and to a lesser extent in female mice. Ear marking significantly changed minute volume compared with handling but not restraint in male mice and in the opposite manner in female mice. Tail vein blood sampling changed minute volume in a significant manner compared with restraint but not compared with handling in both sexes. Retroorbital blood sampling significantly changed minute volume compared with values for both handling and restraint in male mice but only compared with handling in female mice. Tail clipping modified minute volume significantly compared with handling in male mice and compared with restraint in both sexes. Analysis of data showed that routine procedures affect minute volume in mice depending on invasiveness of maneuver and in a sex-biased manner for as long as 24 h after the procedure. Our experiment shows that procedures performed on laboratory mice can change respiratory parameters and can be investigated by plethysmography.

Assessment of storage effects in liquid preserved boar semen.

Fertility of extended boar semen declines within the first 72 h of storage in vitro. Standard semen assessment, such as motility and membrane integrity, allows detection of lethal damage of spermatozoa. However, conventional sperm assessment often lacks standardization and does not allow identification of sub-lethal changes of sperm quality during the initial 72 h of storage. In the present brief review, recent strategies for quality assessment of liquid preserved boar semen are discussed and basic implications for experiments designed to detect storage effects are given.

Simple and economic colloidal centrifugation protocols may be incorporated into the clinical equine sperm processing procedure.

For many years in human assisted-reproduction procedures there have been special protocols to prepare and improve sperm quality. Colloidal centrifugation (CC) is a useful technique that has been proved to enhance semen quality by selection of the best spermatozoa for different species. Its use is recommended to improve fertility of subfertile stallions but current CC protocols are clinically complicated in the equine sperm processing technique due to economic and technical difficulties. The aim of this study was to determine the optimal processing procedures to adapt the use of a CC product (EquiPure™) in the equine reproduction industry. A total of nineteen ejaculates were collected from 10 Purebred Spanish Horses (P.R.E horses) using a Missouri artificial vagina. Gel-free semen aliquots were analyzed prior to treatment (control). Semen was subjected to one of six CC protocols with EquiPure™ and centrifuged samples were statistically evaluated by ANOVA and Duncan tests (p<0.05) for sperm quality and recovery rate. We obtained higher values by colloidal centrifugation in LIN, STR and BCF variables and DNA fragmentation index trended to be lower in most of the CC protocols. The studied protocols were shown to be as efficient in improving equine sperm quality as the current commercial EquiPure™, with the added advantage of being much more economical and simple to use. According to these results it seems to be possible to incorporate single layer and or high colloidal centrifugation volume protocols what would make them simple, economic and clinically viable for the equine sperm processing procedure.

Assessment of diagnostic tools for identifying cattle shedding and super-shedding Escherichia coli O157:H7 in a longitudinal study of naturally infected feedlot steers in Ohio.

The objectives of this study were to compare the performance of different diagnostic protocols (rectoanal mucosal swabs and immunomagnetic separation [RAMS-IMS], fecal samples and IMS [fecal-IMS], and direct plating) to determine the prevalence of Escherichia coli O157:H7 and to evaluate the pattern of E. coli O157:H7 shedding and super-shedding (defined as having a direct plating count equal to or >10(4) colony forming units of E. coli O157:H7 per gram of feces) in a longitudinal study of naturally infected feedlot steers. RAMS and fecal grab samples were obtained at 14-day intervals from 168 Angus-cross beef steers over a period of 22 weeks. Fecal samples were assessed by direct plating and IMS, whereas RAMS were tested only by enrichment followed by IMS to recover E. coli O157:H7. The period prevalence for shedding was high (62%) among feedlot steers and super-shedding was higher (23%) than anticipated. Although direct plating was the least sensitive method to detect E. coli O157:H7-positive samples, over 20% of high bacterial load samples were not detected by RAMS-IMS and/or fecal-IMS. The sensitivity of RAMS-IMS, fecal-IMS, and direct plating protocols was estimated using simple and multilevel mixed-effects logistic regression models, in which the dependent variable was the dichotomous results of each test and gold standard (i.e., parallel interpretation of the three protocols)-positive individuals were included as an independent variable along with other factors such as dietary supplements, time of sampling, and being exposed to a super-shedding pen-mate. The associations between these factors and the sensitivity of the diagnostic protocols were not statistically significant. In conclusion, differences in the reported impact of diet and probiotics on the shedding of E. coli O157:H7 in previous studies using RAMS-IMS or fecal-IMS were unlikely due to their impact on test performance.

Identifying non-sperm particles during flow cytometric physiological assessment: a simple approach.

Flow cytometry is now being used more frequently to determine sperm functional characteristics during semen assessment for artificial insemination. With this methodology, viable and potentially functional cells are detected as unstained events differentiated from non-sperm events through their light-scattering characteristics. However, it can be shown mathematically that identification of sperm on the basis of light scatter leads to significant overestimation of unstained viable cells and underestimation of responding cells in tests of sperm function (subpopulations expressing different fluorescence patterns). We have developed a simple and cost-efficient flow cytometric approach for identifying non-sperm particles that can be carried out in parallel with functional assessments. Our method is based on the sperm's osmotic intolerance. Diluted in water, lethal osmotic shock causes major damage to the cell membranes, and all sperm will stain with propidium iodide (PI). Particulate material which is not PI-positive can then be quantitatively evaluated by FACS analysis and the results substituted in mathematical equations to provide true values for sperm counts and subpopulations. In practical tests, the percentage of non-sperm particles determined by this technique was closely comparable to the figure obtained either by SYBR14/PI staining or by PI/CFDA staining. As well as being valuable with respect to tests of sperm function, the procedure is also suitable for obtaining accurate sperm counts during routine semen evaluation.

Effect of tissue processing on assessment of endoscopic intestinal biopsies in dogs and cats.

BACKGROUND: Prior studies failed to detect significant association between hypoalbuminemia and small intestinal lesions. HYPOTHESIS: Use of pictorial templates will enhance consistency of interpathologist interpretation and identification of intestinal lesions associated with hypoalbuminemia. ANIMALS: Tissues from 62 dogs and 25 cats examined as clinical cases at 7 referral veterinary practices in 4 countries. METHODS: Retrospective, observational study. Histopathology slides from sequential cases undergoing endoscopic biopsy were examined by 4 pathologists by pictorial templates. Changes for 9 microscopic features were recorded as normal, mild, moderate or severe, and 2- and 4-point scales were tested for consistency of interpretation. Logistic regression models determined odds ratios (OR) of histologic lesions being associated with hypoalbuminemia while kappa statistics determined agreement between pathologists on histologic lesions. RESULTS: There was poor agreement (kappa = -0.013 to 0.3) between pathologists, and institution of origin of slides had effect (kappa = 1.0 for 3 of 4 lesions on slides from Institution 5) on agreement between pathologists on selected histologic features. Using 2 point as opposed to 4-point grading scale increased agreement between pathologists (maximum kappa = 0.69 using 4-point scale versus maximum kappa = 1.0 using 2-point scale). Significant association (P = .019- .04; 95% OR = 3.14-10.84) between lacteal dilation and hypoalbuminemia was found by 3 pathologists. CONCLUSIONS AND CLINICAL IMPORTANCE: Substantial inconsistency between pathologists remains despite use of pictorial template because of differences in slide processing. Distinguishing between mild and moderate lesions might be important source of the disagreement among pathologists.

Assessment of prothrombin time, activated partial thromboplastin time, and fibrinogen concentration on equine plasma samples following different storage conditions.

The aim of the present study was to assess the effect of different storage conditions on prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen concentration in clinical samples from healthy horses. A total of 100 healthy horses of varying breeds and gender, ranging in age from 4 to 18 years, with a mean body weight of 480 +/- 70 kg, were used. Blood was collected by jugular venipuncture, and a hemochrome-cytometric examination was conducted on all samples. All blood samples were centrifuged and divided into 4 different aliquots to assess clotting parameters by means of a coagulometer. The first aliquots were analyzed 1 hr after collection, the second aliquots were refrigerated at 8 degrees C for 6 hr, the third aliquots were frozen at -20 degrees C for 24 hr, and the fourth aliquots were frozen at -20 degrees C for 48 hr. Significant differences (P < 0.05) were determined by one-way analysis of variance with repeated measures, and statistical analysis showed a significant effect of the experimental conditions on all parameters studied. In particular, the results demonstrated that coagulation tests can be done within 6 hr when samples are stored at 8 degrees C because the short-term refrigeration does not change the result of analyses; storage at -20 degrees C is acceptable only after 24 hr for PT, aPTT, and fibrinogen measurements because after 48 hr, freezing alters the values of clotting parameters. Therefore, the results of this investigation indicate that clotting parameters remain stable only up to 24 hr in horses without adversely affecting hemostasis test results.

Assessment of canine oocyte viability after transportation and storage under different conditions.

To improve assisted reproductive technologies in the domestic dog, different transport treatments were evaluated for their ability to maintain viability of canine oocytes, as assessed by esterase activity 8h after storage or after 48 h of in vitro maturation (IVM) culture. In Experiment 1, ovaries were transported within reproductive tracts or were excised and stored at either 20 or 37 degrees C in phosphate buffered saline. Oocytes collected from reproductive tracts transported at 37 degrees C had the greatest viability after storage (P<0.05). However, after IVM there were no significant differences among any of the four storage conditions in oocyte viability or meiotic resumption (P=0.05). In Experiment 2, isolated oocytes were transported in either TCM-199 with Hank's salts and Hepes buffer or in TL-Hepes at either 20 or 37 degrees C, or in maturation medium equilibrated with 5% CO(2) at 37 degrees C. In Experiment 2, oocytes transported in Hepes buffered media at 37 degrees C had greater viability rates after storage than did those transported in these same media at 20 degrees C or in sodium bicarbonate buffered medium at 37 degrees C (P<0.001). After IVM, oocytes transported in the 37 degrees C treatment groups had greater viability rates than did those transported at 20 degrees C (P<0.01). Overall, isolated oocytes transported at 37 degrees C had greater rates of meiotic resumption than did those transported at 20 degrees C (P<0.05). Taken together, these data indicate that canine oocytes exhibited sensitivity to lesser temperatures and maintained greater rates of viability during transport at 37 degrees C. Isolated oocytes maintained greater viability than oocytes transported in situ. Hepes buffered media increased viability rates for isolated oocytes transported at 37 degrees C compared to a similar medium buffered with sodium bicarbonate.

[Nonspecific clinical signs in pigs and use of exclusion diagnosis for classical swine fever: a survey among pig farmers and veterinary practitioners]. / Toepassing van uitsluitdiagnostiek voor klassieke varkenspest bij aspecifieke klinische problemen op varkensbedrijven: een enquête onder varkenshouders en dierenartsen.

Outbreaks of Classical Swine Fever (CSF) occurred in spring 2006 in Germany close to the Dutch border. On 6th April Dutch pig farmers were given the possibility to submit blood samples directly via their veterinary practitioner to the National Reference Laboratory for CSF if their pigs had non-specific clinical symptoms or if pigs were being treated with antibiotics. The pig farm was not quarantined and was not visited by the veterinary authorities. Over a period of 9 weeks 156 pig farmers submitted whole blood samples via 50 different veterinary practices. All samples tested negative in the PCR test. These pig farmers and veterinary practitioners were asked to respond to a postal questionnaire with questions regarding their experience with this new diagnostic possibility, the distribution of the costs involved, a comparison with other instruments, such as official notification or use of a leukocyte count test, and their knowledge of clinical signs of CSF. 65 pig farmers (42%) and 33 veterinary practices (66%) returned the questionnaire. The main results indicated that pig farmers (72%) would use this type of exclusion diagnostics sooner than that they would approach the veterinary authorities (practitioners: 86%). Moreover the respondents considered the fact that the farm was not quarantined immediately to be an advantage (pig farmers, 79%; practitioners, 88%). 32 percent of the pig farmers were not aware that they were required to submit blood samples if pigs were being treated with antibiotics (practitioners: 11%). The majority of pig farmers and practitioners were not satisfied with the current distribution of the costs involved: in their opinion the costs of the PCR test, the costs of the veterinary practitioner and the costs for shipping the samples to the reference laboratory should be paid out of the Animal Health Fund (50% government and 50% industry) or by the government. If the current distribution of the costs is not changed, a large proportion of the pig farmers indicated that they would not use this form of exclusion diagnostics for CSF in the future. Pig farmers appeared to have a rather limited knowledge of the clinical signs of CSF: 33% of the pig farmers could mention maximally three clinical signs of CSF, and 7% could not mention a single clinical sign of CSF and said they were entirely dependent on the practitioners' ability to judge a CSF-suspect situation.

Use of a new computer-assisted sperm analyzer for the assessment of motility and viability of dog spermatozoa and evaluation of four different semen extenders for predilution.

The aims of this study were to evaluate a new computer-assisted sperm analyzer (CASA; SpermVision, Minitüb, G) for the analysis of canine spermatozoa, and to suggest suitable semen diluents that should influence motility and viability parameters to the lowest extent. For these purposes, the sperm-rich fractions from 40 ejaculates were diluted with either saline (S), PBS, autologous prostate secretion (third fraction, PROST) or a modified TRIS buffer (TRIS; [Pena, A., Johannisson, A., Linde-Forsberg, C., 1999. Post-thaw evaluation of dog spermatozoa using new triple fluorescent staining and flow cytometry. Theriogenology 52, 965-980]). Viability was investigated both objectively with SYBR-14/PI in SpermVision and subjectively with carboxyfluoresceindiacetate (CFDA) fluorescence stains by optical microscopy. For all diluents and for most parameters, the intra- and inter-assay coefficients of variation were below 10% and 20%. Effects after dilution with TRIS and PROST were similar, but only after dilution with TRIS, total motility significantly correlated with estimated motility in raw semen (P<0.01, R=+0.514). After dilution, estimated and measured motility values were positively related, but correlated significantly with PBS only (P<0.01). After dilution with PBS and S, all motility parameters were significantly lower in comparison with PROST and TRIS, and after dilution with PBS, significantly more membrane damages occurred than with TRIS, S and PROST (P<0.05, <0.05 and <0.01). In samples diluted with TRIS, membrane integrity of spermatozoa determined with SYBR-14/PI and CFDA staining correlated significantly (R=+0.686; P<0.01). Concerning the location of membrane damages, no differences appeared after dilution with different media. In conclusion, SpermVision proved to be an accurate analyzer for the objective assessment of both motility and viability of canine spermatozoa. TRIS and PROST were the media with the lowest influences on parameters of canine semen, and are therefore recommendable for the use in SpermVision.

Batchwise assessment of porcine embryos for cryotolerance.

The viability or developmental ability of porcine embryos after slow-freezing and thawing differs depending on the embryonic stage or the batch, which is defined as a group of embryos obtained from one donor at one time. We froze porcine blastocysts in batches and assessed their cryotolerance by using two expanded blastocysts (EBs) as samples to predict the developmental potential of other blastocysts from the same batch at different stages. Two EBs from the same batch that had been separately frozen were thawed and cultured in vitro for 48 h to examine their in vitro ability to develop to the hatched blastocyst stage. Thereafter, each batch was assigned to Grade A, B, or C according to the viability of the two EBs, i.e., 100% viability (2/2: number of hatched blastocysts/number of cultured EBs) was Grade A; 50% (1/2) was Grade B; and 0% (0/2) was Grade C. The viability of EBs after freeze-thawing and in vitro culture varied depending on the batch and was lower (31.0+/-10.2%, mean+/-S.E.M.; P<0.01) than that of unfrozen controls (96.8+/-2.3%). The viability of frozen-thawed hatched blastocysts (HBs) did not differ among the graded batches, but the blastocyst diameter decreased (from 409 to 326 microm) as the batch grade decreased (from A to C). When both EBs and HBs from batches of the same grade were transferred to recipients (average 11.7 EBs and 16.0 HBs per recipient), the rate of pregnancy and farrowing in recipients decreased (from 77.8% to 0%) and the number of piglets obtained decreased (from 15.3 to 0) as the batch grade decreased. However, when not only frozen-thawed EBs from Grade B or C batches, but also four helper embryos at the morula to early blastocyst stage (which were expected to support the pregnancy) were transferred, the number of piglets generated was higher from EBs from Grade B batches (16.0) than from EBs from Grade C batches (0.0). When frozen-thawed HBs and helper embryos were transferred, the number of piglets generated was higher from HBs from Grade B batches (12.7) than that from HBs from Grade C batches (1.9). After slow-freezing of porcine blastocysts, their rate of survival to the piglet stage differs batchwise, and in vitro viability assessment of sample EBs after freezing and thawing may help in assessing the post-freezing and post-thawing developmental potential of other blastocysts at different stages from the same batch.

Assessment of the repeatability of a milk Ostertagia ostertagi ELISA and effects of sample preparation.

An indirect Ostertagia ostertagi ELISA on milk is a promising diagnostic tool in bovine parasitology. Interpretation of the test results requires a good knowledge of the test characteristics. In this study, border effects, the repeatability of the ELISA and the effect of different factors such as storage, skimming and freeze-thaw cycles of the milk samples were investigated. The border effects trial showed that significant border effects can occur. The repeatability trial was conducted over 3 days. An alternative graphical technique to assess the repeatability over a large number of ELISA plates measured over different days was developed. From these graphs, it was obvious that the ODR values obtained on the third day were deviating from the values on the first and second day. On the third day, also abnormal control values were observed. When the control values were normal, 94% of the variability was explained by the milk sample and 6% by assay variability. The expected 95% range of the difference of 2 ODR readings of the same sample on the same plate and the same sample on different plates was -0.14 to 0.14 and -0.16 to 0.16. No extra variability was observed when samples were tested on a different day, however these results are based on the measurement of 2 days. Storage for 2-4 days at 4 degrees C, using whole milk instead of skimmed milk and up to 2 extra freeze-thaw cycles of the milk samples did not significantly affect the test results.