F. Schmoll

Artificial insemination in cattle with DNA‐treated sperm

Abstract We have investigated the use of sperm cells as vectors for transferring exogenous DNA into the genome of cattle by artificial insemination with DNA‐treated sperm. First we demonstrated the DNA‐binding ability of cattle sperm with radioactively labeled DNA. For artificial insemination ejaculated semen was washed and incubated with 1 μg DNA/106 sperm for one hour at 37°C. Three hundred synchronized heifers were inseminated once with a dose of 40×106 sperm. Forty‐five calves and 41 fetuses were obtained. Southern analysis revealed in one calf a signal after probing with the 1 kb Pst I fragment of pSV2‐cat.

Effects of different cryoprotectants and carbohydrates on freezing of matured and unmatured bovine oocytes

Cumulus cell-enclosed bovine oocytes in germinal vesicle (GV) and in metaphase II (MII) stages were cryopreserved. Different concentrations (1 M; 1.5 M) of various cryoprotectants (glycerol, PROH, DMSO) were tested. After thawing, the oocytes were exposed to various carbohydrates (sucrose, lactose, trehalose) at a concentration of 0.1 M and 0.25 M for cryoprotectant removal. Developmental capacity of the frozen-thawed oocytes was studied by in vitro maturation, fertilization and culture. We found no difference in subsequent development using glycerol or PROH for GV and MII oocytes. The DMSO treatment led to significantly better cleavage and development up to 4-cell stage in MII oocytes. Development beyond the 8-cell stage was obtained only when unmatured oocytes were frozen. No difference in the efficiency of the 3 cryoprotectants was detected in MII oocytes. However, in GV oocytes, glycerol and PROH yielded significantly better cleavage and 4-cell rate compared to DMSO (P<0.001). Influence of the concentration of a cryoprotectant on development was not observed in GV or MII oocytes. Among the 3 cryoprotectants, DMSO was less suitable, at both concentrations, than PROH and glycerol for the development of 6- to 8-cell stage embryos in the GV group. In the MII group, 1.5 M DMSO was as efficient as PROH and as glycerol at a 1.5-M concentration, and it was more efficient than 1 M glycerol. The use of carbohydrates during rehydration did not render a beneficial effect at either of the 2 concentrations, and when no carbohydrates were used in the MII group the oocytes cleaved better than GV oocytes.

The use of endogenous and exogenous reference RNAs for qualitative and quantitative detection of PRRSV in porcine semen

Abstract Semen is known to be a route of porcine reproductive and respiratory syndrome virus (PRRSV) transmission. A method was developed for qualitative and quantitative detection of the seminal cell-associated PRRSV RNA in relation to endogenous and exogenous reference RNAs. As endogenous control for one-step real-time reverse transcription (RT)-PCR UBE2D2 mRNA was selected. Particularly for the analysis of persistent infections associated with low copy numbers of PRRSV RNA, UBE2D2 mRNA is an ideal control due to its low expression in seminal cells and its detection in all samples analysed (n =36). However, the amount of UBE2D2 mRNA in porcine semen varied (up to 106-fold), thus its use is limited to qualitative detection of PRRSV RNA. For quantitation, a synthetic, non-metazoan RNA was added to the RNA isolation reaction at an exact copy number. The photosynthesis gene ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) from Arabidopsis thaliana was used as an exogenous spike. Unexpectedly, PRRSV RNA was detected in a herd of specific pathogen-free (SPF) boars which were tested ELISA-negative for anti-PRRSV antibodies. Therefore, RT-PCR for seminal cell-associated PRRSV is a powerful tool for managing the SPF status during quarantine programs and for routine outbreak investigations.

Parameters of humoral and cellular immunity following vaccination of pigs with a European modified-live strain of porcine reproductive and respiratory syndrome virus (PRRSV).

Parameters of humoral and cellular immunity were investigated in pigs experimentally infected with a modified-live European porcine reproductive and respiratory syndrome virus (PRRSV, strain DV). PRRSV was detected by real-time RT-PCR and PRRSV-specific antibodies by a commercial ELISA test-kit, respectively. Interleukins IL-1alpha, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF-alpha) and interferon-gamma (IFN-gamma) as well as IL-2 receptor (IL-2R) were quantified at mRNA level using RT-PCR. Subpopulations of blood lymphocytes were assayed using flow cytometry. No significant changes neither in cytokine expression nor in shifts of CD4 and CD8 markers could be found, but similar curve diagrams concerning CD8 single positive T cells could be observed in all vaccinated animals with an initial decrease and an increase between post-infection days (PIDs) 7 and 14. In the vaccination group, TNF-alpha and IL-6 tended to be increased at PIDs 22 and 40, whereas no increase could be seen in IFN-gamma. When comparing the in vivo immune response to that being seen in in vitro experiments, similar shifts of CD4/CD8 lymphocyte subpopulations may be seen. Cytokine curve diagrams, however, do not reflect the in vitro findings to that extent.

A Two-Years' Survey on the Prevalence of Tuberculosis Caused by Mycobacterium caprae in Red Deer (Cervus elaphus) in the Tyrol, Austria

A survey of 143 hunter-harvested red deer for tuberculosis was conducted in an Alpine area in Western Austria over two subsequent years. There, single tuberculosis cases caused by Mycobacterium caprae had been detected in cattle and red deer over the preceding decade. The area under investigation covered approximately 500 km2, divided into five different hunting plots. Lymph nodes of red deer were examined grossly and microscopically for typical tuberculosis-like lesions and additionally by microbiological culturing. Executing a detailed hunting plan, nine M. caprae isolates were obtained. Six out of nine originated from one single hunting plot with the highest estimated prevalence of tuberculosis, that is, 23.1%. All isolates were genotyped by mycobacterial interspersed repetitive unit—variable number of tandem repeat (MIRU-VNTR) typing of 24 standard loci plus VNTR 1982. All nine isolates belonged to a single cluster termed “Lechtal” which had been found in cattle and red deer in the region, demonstrating a remarkable dominance and stability over ten years. This is the first report on a systematic prospective study investigating the prevalence and strain variability of M. caprae infection in red deer in Austria and in the Alpine countries.

Detection and molecular characterization of Suid herpesvirus type 1 in Austrian wild boar and hunting dogs.

Aujeszkys disease (AD), caused by Suid herpesvirus type 1 (SuHV-1), is an economically important disease in domestic swine. Thus, rigorous control programmes have been implemented and consecutively AD in domestic swine was successfully eradicated in many countries, including Austria. However, SuHV-1 continues to thrive in wild boar populations, as indicated by high seroprevalences in a number of European countries and by occasional cases of AD in hunting dogs. For the first time, SuHV-1 was detected in Austrian wild boar and a molecular characterization of SuHV-1 isolated from wild boar and hunting dogs was performed. Results of preliminary serological analyses suggest a regional SuHV-1 seroprevalence of over 30% in free-living and almost 70% in fenced wild boar from Eastern Austria. Molecular typing of Austrian SuHV-1 isolates of wild boar origin revealed the presence of two genetically distinct variants of SuHV-1, both capable of infecting dogs that have been exposed to infected wild boar during hunting.

Variation of the growth hormone gene in ryr 1 genotyped Austrian pig breeds.

SUMMARY Polymorphism in the second intron of the porcine growth hormone gene of 273 Austrian Landrace and 81 Austrian Edelschwein pigs was investigated with a PCR-RFLP-technique. Results showed significantly different genotype patterns between the two breeds. The frequency of the Hae II(-) allele was significantly (P < 0,001) higher in the landrace than in the Edelschwein population (0,83 and 0,47 resp.). The Msp I(+) allele was predominant in both breeds but signifanctly higher in the Landrace (0,98 versus 0,69; P < 0,01). Analyses the Hae II/Msp I locus combination revealed also in breed specific difference. In the Landrace a very low interaction was found between the Hae II and ryr 1 locus, and between Msp I and ryr 1 locus (c. c. = 0,181 and 0,186 resp.). The correlation was slightly stronger (c. c. = 0,266) between the ryr 1 and Hae II/Msp I genotypes. No correlation was detected among the three loci in the Edelschwein population. ZUSAMMENFASSUNG: Variabilität des Somatotropin Gens in österreichischen Schweinerassen genotypisiert hinsicbtlich ryr 1 An 273 Österreichischen Landrasse Schweinen und 81 Österreichischen Edelschweinen wurde der Polymorphismus am zweiten Intron des Schweinewachstumshormons mittels PCR-RFLP-Technik untersucht. Genotypen-und Genfrequenzen waren zwischen den beiden Rassen signifikant verschie- den. Die Hae II(-) Allelfrequenz war bei den Landrassetieren signifikant höher (P < 0,001) als bei den Edelschweinen (0,83 bzw. 0,47). In beiden Rassen überwiegte das Msp I(+) Allel, das aber signifikant öfters bei der Landrasse auftrat (0,98 bzw. 0,69; P < 0,01). Die Verteilung der Locuskombination von Hae II/Msp I zwischen den beiden Rassen war ebenfalls unterschiedlich. Bei der Landrasse konnte nur ein sehr geringer Zusammenhang zwischen dem Hae II und ryr 1 Locus (c. c. = 0,181 bzw. 0,186) und auch zwischen dem Msp I und ryr 1 Locus festgestellt werden. Die Korrelation zwischen dem ryr 1 und Hae II/Msp I Genotyp war geringfügig größ;er (c. c. = 0,266). Bei den Edelschweinen konnte kein Zusammenhang zwischen den drei untersuchten Loci festgestellt werden.

Comparison of aggregation and injection techniques in producing chimeras with embryonic stem cells in mice

We analyzed embryonic stem cell lines for their capacity to produce aggregation chimeras with diploid or developmentally compromised tetraploid embryos. Descendants of embryonic stem cells which contributed to midgestation fetuses at high levels were capable of supporting fetal development also with tetraploid partners. Different numbers of embryonic stem cells were introduced into diploid and tetraploid morulae as well as into blastocysts by microinjection. There were no differences in the frequency of embryonic stem cell-containing fetuses when comparing aggregation or injection into morulae versus blastocysts. However, the distribution pattern of embryonic stem cell derivatives in chimeric fetuses suggested that pre-compaction embryos are more suitable for generating fetuses with high embryonic stem cell contribution. Injection of embryonic stem cells into tetraploid embryos showed that completely embryonic stem cell-derived fetuses can also be produced by this technique. Totally embryonic stem cell derived fetuses were observed in each group, when embryonic stem cells were injected into diploid embryos. However, the rate of chimeras and chimerism was lower when 1 or 3 embryonic stem cells were used versus 8 or 15 cells. This suggests that the number of embryonic stem cells introduced might play a role in the colonization ability.

Effects on boar semen quality after infection with porcine reproductive and respiratory syndrome virus: a case report

The effect of porcine reproductive and respiratory syndrome virus (PRRSV) on semen quality was examined in a group of 11 spontaneously infected boars in a commercial boar stud. Semen samples were collected 4 weeks prior to 4 weeks post-infection (wpi). Infection with PRRSV of the European genotype subtype 1 (EU-1) was verified by specific quantitative real-time polymerase chain reaction (RT-PCR) in 36% of the serum samples. All boars seroconverted before 4 wpi and remained in normal condition throughout the study. Comparison of the percentage of morphologically intact spermatozoa revealed an increase of acrosome-defective spermatozoa (P = 0.012) between −4 and 4 wpi. Significant deleterious effects on semen quality were detected for membrane integrity when semen had been stored for 2 days after sampling. Analysis of sperm subpopulations in a thermoresistance test on day 7 after sampling revealed alterations in the percentage of circular, progressively motile spermatozoa (P = 0.013), in the percentage of non-linear, progressively motile spermatozoa (P = 0.01), and on the amplitude of lateral sperm head displacement (P = 0.047). There was no difference in the incidence of mitochondrially active spermatozoa (P = 0.075). Investigation of routine production data between pre- and post-infection status showed no differences on ejaculate volume (P = 0.417), sperm concentration (P = 0.788), and percentage of motile spermatozoa (P = 0.321). This case report provides insights into a potential control strategy for PRRSV outbreaks in boar studs.

Correlation between antibodies against porcine reproductive and respiratory syndrome virus and pathological-anatomical organ findings in slaughter pigs at farm level

OBJECTIVE The porcine reproductive and respiratory syndrome (PRRS) worldwide causes important economic losses in pig production. Its causative agent, the PRRS virus (PRRSV), is one of the most frequently detected infectious agents in relation to respiratory diseases in pigs in Austria. We investigated the correlation between the PRRSV status of pig farms, determined by detection of PRRSV antibodies in the serum of slaughter pigs, and the prevalence of pathological-anatomical lung lesions in slaughter pigs of the respective farms. MATERIAL AND METHODS Between December 1, 2011 and April 16, 2012, a total of 1056 serum samples of slaughter pigs from 66 pig farms were collected at an Austrian abattoir. The presence of PRRSV antibodies was tested by enzyme-linked immunosorbent assay in each sample and the PRRSV status of the respective farms was determined. No PRRSV vaccination was performed on any of the farms. In addition, the pathological-anatomical lung lesions of all slaughter pigs of the 66 farms that were slaughtered between September 1, 2011 and December 11, 2012 were recorded by authorized veterinarians at the abattoir. The prevalence of lung lesions and pleuritis in PRRSV-positive and unsuspected farms was compared and statistically interpreted. RESULTS Slaughter pigs of PRRSV positive farms had a significantly higher prevalence of severe lung lesions and pleuritis visceralis and parietalis than slaughter pigs of PRRSV unsuspected farms. Pigs of combined farms (nursery and fattening unit at the same location) displayed a tendency for more moderate and severe lung lesions than pigs of exclusive fattening farms. CONCLUSIONS AND CLINICAL RELEVANCE In the present study, the PRRSV status of pig farms displayed a significant influence on the prevalence of lung lesions in the slaughter pigs. Findings untypical for PRRS, including pleuritis, were also found significantly more often on those farms. This leads to the conclusion that other primary and/or secondary infections are involved, which can be exacerbated by the immunosuppressive effect of the PRRSV. There was a tendency for combined farms to be more severely affected than fattening farms.