O. Szenci

Early pregnancy diagnosis in cattle by means of linear-array real-time ultrasound scanning of the uterus and a qualitative and quantitative milk progesterone test.

We compared three methods for diagnosing early pregnancy in cattle: 1) a trans-rectal ultrasound scan of the uterus, 2) a cow-side enzymeimmunoassay (EIA) milk progesterone test 3) a radioimmunoassay (RIA) milk progesterone test. Scanning of the uterus was performed in 148 cows. These cows were not detected in estrus before scanning, which took place between Days 21 and 33 after insemination (AI). A considerable difference was noted between the reliability of the scannings performed at an early stage (Days 21 to 25) and those performed at a later stage (Days 26 to 33). The sensitivity and specificity of the ultrasound examination between Days 21 and 25 were only 44.8% and 82.3%, respectively, but were 97.7% and 87.8% between Days 26 and 33, respectively. Milk samples were collected on the day of AI. (Day 0) and 21 days later. Samples that were positive in the EIA test always contained more than 1 ng/ml progesterone (P4); however, 20% of the negative EIA samples contained also more than 1 ng/ml P4. Only 59% of the animals showing a negative EIA test on Day 0 and a positive test on Day 21, indicating pregnancy, calved, while 16% of the cows with a negative test on Day 0 and Day 21, indicating nonpregnancy, turned out to be pregnant. Of the 82 animals with P4 levels lower than 1 ng/ml on Day 0 and higher than 1 ng/ml on Day 21, only 61.0% calved. All 14 cows with low levels both on Day 0 and Day 21, indicating nonpregnancy, were found to be not pregnant. The influence of both early embryonic death and the accumulation of intrauterine fluids on the accuracy of these tests are discussed.

Early pregnancy diagnosis in sheep by progesterone and pregnancy-associated glycoprotein tests

The aim of this study was to compare the accuracy of the progesterone (P4) and pregnancy associated glycoprotein (PAG) tests for determination of early pregnancy in sheep. Estrus was synchronized in 182 Awassi x Merino ewes and blood samples were collected at Days 0 (day of the insemination), 18, 22, 29, 36, and 50 after artificial insemination (AI). Plasma P4 concentrations at Days 0 and 18 were determined by double antibody radioimmunoassay, while PAG concentrations at Days 22, 29, 36 and 50 were determined by a heterologous, double-antibody radioimmunoassay (RIA) using the bovine PAG 67 kDa subunit as tracer and standard and rabbit antiserum raised against a mixture of caprine 55 and 59 kDa PAG subunits as the first antibody. The discriminatory value for diagnosis of pregnancy by the P4 and the PAG-RIA tests was > or = 1 ng/ml. Based on lambing data, the accuracy for diagnosing pregnant (sensitivity) and non-pregnant ewes (specificity) and predictivity of both tests were calculated. The sensitivity, specificity, positive and negative predictive values for P4 and PAG tests were 100, 95.4, 81.6, and 100% at Day 18 (P4) and 93.5, 100, 100 and 98.7% at Day 22 (PAG), respectively. For diagnosis of non-pregnant ewes the PAG test had significantly higher specificity than the P4 test (P < 0.05). It is concluded that ovine pregnancy can be reliably diagnosed at Day 22 after AI by using a heterologous radioimmunoassay of PAG.

Evaluation of false ultrasonographic diagnoses in cows by measuring plasma levels of bovine pregnancy-associated glycoprotein 1

Between days 27 and 59 after artificial insemination (AI), 189 ultrasonographic pregnancy diagnoses were made in 56 dairy cows using a 7.5 MHz linear-array rectal transducer. Blood samples were withdrawn from a jugular vein on the day of Al, on day 21, and after each ultrasonographic examination between days 27 and 31, days 34 and 38, days 41 and 45 and days 55 and 59 after AI. Plasma concentrations of bovine pregnancy- associated glycoprotein 1 (bPAG-1) were measured by radioimmunoassay. The results showed that before day 31, ultrasonographic scanning was not very sensitive because six of the 30 calving cows were incorrectly diagnosed as non-pregnant. In five of these animals, the uterus was located far cranial to the pelvic inlet. Five of the cows examined between days 27 and 31 were pregnant on the basis of plasma bPAG-1 levels on the same day, using 0.5 ng/ml as the cut-off point. Plasma levels of bPAG-1 and progesterone proved that four of the cows which had early positive ultrasonographic diagnoses but did not produce a calf, were pregnant when they were examined.

Effect of induction of late embryonic mortality on plasma profiles of pregnancy associated glycoprotein 1 in heifers.

Inoculation with Actinomyces pyogenes and administration of prostaglandin (PG)F(2alpha) were used to induce late embryonic mortality (LEM) in heifers (n=8) on Days 30-38 of pregnancy in order to compare the profile for bovine pregnancy associated glycoprotein 1 (PAG1), progesterone and 15-keto-13,14-dihydro-PGF(2alpha) (PGFM). Two pregnant heifers were used as controls. Inoculation into the uterine body caused LEM, as established by ultrasonography in each heifer within 24h of treatment. When the inoculum was injected into the first part of the cervix, LEM occurred in one of two heifers (Heifer A) between 48 and 72 h after treatment. Similarly, PGF(2alpha) treatment caused LEM in three of four heifers. In six of eight heifers, PAG1 started to decrease steadily when it was accompanied by the subsequent death of the embryo. Inoculation through the cervix caused luteolysis in three of four heifers within 6-10 days after induction. After induction of LEM, PGFM concentrations showed a two to 3.8 fold increase in three of four heifers during the following six days, and from that time changed within normal ranges. The results of this study indicate that a PAG1 assay may provide an alternative method to ultrasonography for determining LEM in the cow.

Equine encephalomyelitis outbreak caused by a genetic lineage 2 West Nile Virus in Hungary

BACKGROUND The spread of lineage 2 West Nile virus (WNV) from sub-Saharan regions to Europe and the unpredictable change in pathogenicity indicate a potential public and veterinary health threat and requires scientific awareness. OBJECTIVES To describe the results of clinical and virological investigations of the 1st outbreak of a genetic lineage 2 WNV encephalomyelitis in horses. ANIMALS Seventeen horses with neurologic signs. METHODS Information regarding signalment, clinical signs, and outcome was obtained for each animal. Serology was performed in 15 cases, clinicopathological examination in 7 cases, and cerebrospinal fluid was collected from 2 horses. Histopathology was carried out in 4 horses, 2 of which were assessed for the presence of WNV in their nervous system. RESULTS WNV neutralizing antibody titers were between 10 and 270 (median, 90) and the results of other serological assays were in agreement with those of the plaque reduction neutralization test. Common signs included ataxia, weakness, asymmetric gait, muscle tremors, hypersensitivity, cranial nerve deficits, and recumbency. Twelve animals survived. Amplicons derived from the infection-positive specimens allowed molecular characterization of the viral strain. CONCLUSIONS AND CLINICAL IMPORTANCE From our results, we conclude that this outbreak was caused by a lineage 2 WNV strain, even though such strains often are considered nonpathogenic. Neurological signs and survival rates were similar to those reported for lineage 1 virus infections. The disease occurrence was not geographically limited as had been the typical case during European outbreaks; this report describes a substantial northwestern spread of the pathogen.

Effect of uterus position relative to the pelvic inlet on the accuracy of early bovine pregnancy diagnosis by means of ultrasonography.

A 5-MHz sector transducer (experiment 1) and a 7.5 MHz linear-array transducer (experiment 2) were used for early pregnancy diagnosis in two Hungarian dairy herds under field conditions. In the first experiment, ultrasound scanning was conducted three times at 3 or 4-day intervals between 24 and 33 days after artificial insemination (AI), while in the second experiment, it was performed two times at 7-day intervals between 27 and 38 days post-insemination. Significantly more incorrect non-pregnancy diagnoses were made between 24 and 33 days (first experiment) and between 27 and 38 days (second experiment) after AI in cows in which the uterus was located far cranial to the pelvic inlet than in animals in which the uterus was located within or close to the pelvic inlet at the first ultrasonographic examination.

Characterisation of pregnancy losses after embryo transfer by measuring plasma progesterone and bovine pregnancy-associated glycoprotein-1 concentrations

The aim of this analysis was to determine whether pregnancy loss (PL) after embryo transfer (ET) in cattle was related to maternal progesterone (P4) concentrations during and shortly after ET, and maternal bovine pregnancy-associated glycoprotein-1 (bPAG-1) concentrations in plasma at days 25-35 of gestation. Embryos (n=260) were produced either in vivo after superovulation (n=115), or in vitro from oocytes (obtained with ovum pick-up) in co-culture (n=44) or cultured in a synthetic medium (n=101). Overall, PL was 56.9% (148) and no significant differences occurred in calving rate among the three embryo production groups. There was no difference in P4 concentrations on days 7-14 of gestation in the three groups, nor between ongoing and interrupted pregnancies. Between days 25 and 35 of pregnancy, bPAG-1 concentrations were unaffected by embryo production, but in cattle that had PL between days 26 and 120, four bPAG-1 profiles could be detected. Between days 25 and 32, bPAG-1 concentrations were influenced by PL, and concentrations were significantly lower in animals in which PL occurred between days 26 and 120 than in those animals that aborted later or calved at term. Early P4 concentrations suggested that maternal luteal factors were not responsible for PL which appeared to be caused by impaired conceptus development (regardless of embryo type) as reflected by low maternal bPAG-1 concentrations prior to embryonic death.

Stress Preconditioning of Boar Spermatozoa: A New Approach to Enhance Semen Quality*

Semen preparation and cryopreservation require finely adjusted procedures. Gametes are sensitive to environmental stresses, so in vitro procedures aim to minimize the inevitable harmful conditions. Applying stress to precondition cells has only been investigated recently. Studies demonstrated that by utilizing a well defined and properly applied hydrostatic pressure (HP) stress treatment to spermatozoa before in vitro storage, cryopreservation or insemination, cell survival and fertility improved compared with untreated controls. The birth of healthy piglets from treated fresh or frozen-thawed semen demonstrates the in vivo safety of the procedure. Although the biological mechanism is still unclear, several processes incorporating cellular stress response might explain the observations. This paper summarizes results, background, aspects and considerations of HP treatment for porcine semen. The new principle, i.e. to improve the stress tolerance by a defined sublethal stress may outline a new strategy in assisted reproductive technologies with unique theoretical and practical consequences.

Symptomless intrauterine transmission of bovine herpesvirus 4 to bovine fetuses.

Blood samples of 31 healthy calves and their dams taken immediately after calving before colostrum uptake, and at days 11, 23 and 8 weeks, spleens of seven stillborn calves were analysed in order to determine the source and time of bovine herpesvirus type 4 infection. All the calves were born as seronegatives, while all cattle were seropositives. Viral DNA were amplified by a nested PCR assay from 54.8% of peripheral blood leukocyte samples of newborn calves taken before colostrum uptake, and from all cattle and from their colostrums. Real time PCR detected higher virus level in peripheral blood leukocytes in adult cattle, then in their newborn calves. Bovine semen cells (spermatozoa and leukocyte fractions), spleens of stillborn calves also carried viral genomes. Our results prove, that bovine fetuses can be infected in utero by BoHV-4, but are born as seronegatives. After human examples this is the first report in veterinary virology on intrauterine transmission of a herpesvirus without acute consequences. This phenomenon could explain the low antigenicity of BoHV-4 proteins and lack of neutralizating antibodies. BoHV-4, a gammaherpesvirus, could serve as an animal model for studying inapparent herpesviral infections of human fetuses.

Risk assessment of postpartum uterine disease and consequences of puerperal metritis for subsequent metabolic status, reproduction and milk yield in dairy cows

The objective of this study was to determine some metabolic and other factors predicting the risk of postpartum uterine disease (PUD), and the effects of puerperal metritis (PM) on metabolic status, reproduction and milk yield were analysed. A total of 105 Holstein-Friesian cows were included, and sampled on day < -14 prepartum and days 4, 10-14, 28-35 and 56-63 postpartum for metabolic tests. From day 4 the development of PUD, and from days 28-35 the ovarian activity was monitored. When grade > or = 1 + ketonuria was present on day 4 postpartum, this indicated a higher probability of PUD [odds ratio (OR) 2.64; P < 0.05] including PM occurring on days 10-14 (OR: 2.65; P < 0.05). Plasma nonesterified fatty acid (NEFA) concentrations > 0.200 mmol/l on days < -14 prepartum indicated a higher risk of uterine diseases (OR: 3.44; P < 0.05). The odds of PUD increased, depending on whether a body condition score (BCS) loss of > or = 1.0 occurred between days < -14 and 28-35 (OR: 2.82; P < 0.05), between days < -14 and 10-14 (OR: 4.79; P < 0.01) or between days 10-14 and 28-35 (OR: 10.81; P < 0.01). PM was more probable (OR: 27.3; P < 0.001) in cows with retained placenta. The risk of uterine diseases was lower in multiparous than in primiparous cows (OR: 0.29; P < 0.01). PM increased the risk of ovarian inactivity between days 28 and 35 (OR: 2.83; P < 0.05). Cows affected with PM (PM+ cows) showed lower milk production on day 4 (kg; P < 0.05) and lower milk production (P < 0.05), milk fat and milk protein production (kg; P < 0.01; P < 0.01) in the first 100 days of lactation than did PM-cows.