Bovine Fetuses Research Articles

Expression and Distribution of Intermediate‐filament Proteins and Laminin during the Development of the Bovine Müllerian Duct

The expression pattern of several intermediate-filament proteins (vimentin, cytokeratin 8, 18 and 19) and the basal lamina component laminin was investigated in the Wolffian and the Müllerian ducts of bovine embryos and fetuses. The material studied comprised sexually undifferentiated stages [crown-rump length (CRL) 0.9 cm/1.0 cm/1.2 cm/1.9 cm/2.5 cm] and female stages (CRL 3.0 cm/4.2 cm/5.1 cm). Laminin could be demonstrated in the basal lamina of the developing Wolffian and Müllerian duct as well as in the stroma surrounding the Müllerian duct. The intermediate-filament protein vimentin was expressed in the mesothelium of the funnel field and in the epithelium of the Müllerian duct in all studied specimens, whereas the epithelial cells of the Wolffian duct only showed vimentin expression from a CRL of 2.2 cm onwards. In the cranial part of the Müllerian ducts only a few cells stained with pan-cytokeratin antibodies, whereas mesothelium and epithelium of the Wolffian duct showed as distinct immunostaining in all investigated stages. Both genital ducts showed no immunostaining with the antibody against cytokeratin 19 at any time of development. We conclude from our immunohistochemical results that the epithelial cells of the Wollfian duct do not contribute cells to the developing Müllerian duct.

Efeitos de diferentes sistemas de cultivo in vitro sobre o crescimento de folículos pré-antrais isolados de ovários de fetos bovinos

O objetivo deste estudo foi aplicar diferentes sistemas de cultivo in vitro para folículos pré-antrais de fetos bovinos da raça Nelore no último trimestre de gestação e para identificar o sistema mais eficiente durante o crescimento dos folículos isolados. Para isso, folículos pré-antrais foram isolados mecanicamente e submetidos ao cultivo individual, por 9 dias, em meio não suplementado ou suplementado com soro fetal bovino (SFB), albumina sérica bovina (BSA) ou suplemento definido sintético substituto do soro KnockoutSR (KNO). Avaliou-se ainda, o efeito do gel de colágeno ou monocamada de fibroblastos fetais bovinos como substrato para o cultivo in vitro. A avaliação do aumento de diâmetro folicular foi realizada no dia da colheita (0 hora) e a cada 72 horas de cultivo. A associação entre meio suplementado com SFB e uso de gel de colágeno como substrato foram significativamente mais efetivos sobre o aumento do diâmetro folicular quando comparados aos demais tratamentos. Quando fragmentos de tecido ovariano foram submetidos ao cultivo in vitro, não houve preservação da ultraestrutura folicular por mais de 3 dias de cultivo, em qualquer dos tratamentos utilizados. Ainda não está estabelecido um sistema de cultivo adequado que sustente a diferenciação e multiplicação das células da granulosa e que mantenha o contato das mesmas com o oócito para prover moléculas e fatores que supram a demanda metabólica. Entendemos que esta pesquisa apresentou avanços promissores na busca pelo estabelecimento um sistema de cultivo in vitro de folículos pré-antrais em bovinos.

Diagnostic Evidence of Staphylococcus Warneri as a Possible Cause of Bovine Abortion

Following a routine necropsy of a bovine fetus aborted at 5 months of gestation, placenta, fetal tissue samples, and stomach contents were subjected to a number of laboratory tests. Staphylococcus warneri was isolated in pure culture from the lung, liver, and stomach contents, whereas the placenta yielded S. warneri and a number of contaminants. Gross evaluation of agar plates showed predominant colonies to be morphologically consistent with those of S. warneri and the identity of the agent was further confirmed on a Trek Diagnostic Systems Sensititre, gram-positive identification (GPID) plate. Microscopic evaluation of fetal tissue sections showed extensive necrotizing lesions of the tongue, lung, and placenta in which there were numerous coccoid shaped gram-positive bacteria with morphology consistent with Staphylococcus spp. These results provide strong diagnostic evidence of S. warneri as a possible cause of bovine abortion and suggest there should be further investigations into the abortivirulence of this agent.

Monitoring the bovine fetus during stage II of parturition using pulse oximetry

Measurement of oxygen saturation using pulse oximetry is an established method of continuous monitoring of the well-being of the human fetus during parturition. In veterinary medicine, pulse oximetry has been used almost exclusively in intensive care and anesthesiology. The goal of the present study was to investigate the physiological changes in oxygen saturation of the bovine fetus during stage II of parturition and to determine whether the findings can be used to predict postnatal acidosis. The correlation between the oxygen saturation (SpO(2)) measured via pulse oximetry and the oxygen saturation (SaO(2)) of arterial blood measured via blood gas analysis was determined in 23 newborn calves. In addition, the oxygen saturation was monitored continuously via pulse oximetry (FSpO(2)) in 33 bovine fetuses during stage II of parturition. Correlations between the FSpO(2) values during the last 30 and 5min of stage II of parturition and the postpartum values for pH, partial pressures of oxygen and carbon dioxide, bicarbonate concentration, BE, SaO(2) and lactate concentration in arterial blood were determined. There was a high correlation between SpO(2) and SaO(2) postpartum (r=0.923). The FSpO(2) values correlated moderately with the pH and BE and weakly with the lactate concentration postpartum; calves with a pH<7.2, a BE<-3mM/L or a lactate concentration of >5.4mM/L had significantly lower FSpO(2) values than non-acidotic calves. FSpO(2) values <30% for a period of at least 2min had the highest predictive value for a calf born with a pH<7.2. Pulse oximetry is a novel method of monitoring the bovine fetus during parturition; however, technical modifications are required to improve its usefulness.

Histopathological and immunohistochemical aspects of Neospora caninum diagnosis in bovine aborted fetuses

Lesions suggestive of protozoal infection such as nonsuppurative myositis, nonsuppurative myocarditis, nonsuppurative pneumonia associated with focal cellular necrosis and multifocal necrotizing nonsuppurative encephalitis were observed in 89 (34%) out of 258 aborted bovine fetuses. Immunohistochemistry (IHC) revealed tachyzoites and cysts of Neospora caninum in 55 (61.8%) of these 89 fetuses. Several fetal variables were tested by multivariate logistic regression, in which increasing odds for positivity in IHC were associated with the presence of lesion in the lung (OR = 12.11) and less autolytic brain tissue (brain consistency, OR = 5.73). However, autolysis in the brain precluded presumptive diagnosis made by histopathology in 53% of the fetuses examined. These results indicate that, when fetal brain is autolyzed, the lung may be adequately used for the presumptive diagnosis of N. caninum infection. The results also confirmed that, even autolyzed, the brain is the most suitable organ for anti- N. caninum IHC.

First identification of Neospora caninum infection in aborted bovine foetuses in China

The first identification of Neospora caninum infection in the tissues of aborted bovine foetuses in China is reported. Aborted foetuses were collected from 16 dams, and 12 of the dams had high serum antibody titres to N. caninum determined using an ELISA test kit. The Nc-5 gene of N. caninum was amplified from DNA samples extracted from brains of four aborted foetuses using a Neospora-specific PCR assay, confirming N. caninum infection in the aborted foetuses. Histology and immunohistochemistry showed thick-walled (3 μm) tissue cyst in 25 μm diameter in the brain of one foetus. Non-suppurative encephalomyelitis, focal haemorrhage, hepatic lesions consisted of lymphocyte infiltration and haemorrhage were also found in the heart and lung of the foetus. Thus, we have confirmed for the first time the infection of N. caninum in aborted foetuses of cattle in the People's Republic of China.

SOMATIC CELL NUCLEAR TRANSFER USING DONOR CELLS SELECTED FOR DNA METHYLTRANSFERASE 1 (DNMT1) EXPRESSION

Bovine nuclear transfer (NT) embryos and fetuses often display abnormalities that are not observed in their in vivo produced counterparts, and are attributed to large offspring syndrome. Gene expression is altered in NT embryos and is thought to be due to inadequate reprogramming of the somatic cell nucleus. It is known that the embryonic genome must be rapidly demethylated, but often does not occur normally in NT embryos. This may be due to the introduction of Dnmt1s into the NT embryo, the enzyme responsible for maintaining DNA methylation (DM) in somatic cells, but which is never found in early embryos. Chemicals, such as 5azaC, have been used to artificially reduce DM, but have proven problematic in NT. We previously showed that culture without chemicals can be used, with cell type and time in culture affecting expression of Dnmt1 in donor cells. Here we tested the effects of using donor cells selected for Dnmt1 expression (low vs. high) as a means to reduce DM in NT embryos. Cell lines were grown to passage 4, 8 and 12, diluted out and frozen in individual use aliquots for later use. An aliquot of each cell line was taken for RNA isolation, and Dnmt1 expression analysis using Real-time RTPCR. Two cell lines with the same genotype, but at different passages were selected as having higher (HD) and lower Dnmt1 (LD) expression for use in NT experiments. Cumulus oocyte complexes were retrieved by slashing 2–8mm follicles on ovaries obtained from the abattoir, and were matured for 17–19h. Metaphase II oocytes (n=874) were selected for polar body extrusion and were enucleated for use in NT. Cells were transferred from either HD or LD groups, and fused. Fused couplets were activated with ionomycin and DMAP using standard protocols, and cultured within treatment in KSOM to d7 (activation=0). Global DM was evaluated in NT (LD and HD) and in vitro produced (IVP) control embryos (2-cell, 8-cell and blastocyst stages, 10/treatment and stage) by immunocytochemistry using 5MeC antibody (Calbiochem) and a FITC-conjugated secondary antibody (Santa Cruz). IgG and PBS served as negative controls to show antibody specificity. Pixel data were collected using AxioVision software (Zeiss) and were analyzed by LSMeans ANOVA using SAS, and significance was deemed p<0.05. There was significantly less DM in 2-cell IVP when compared to either LD or HD NT embryos (p<0.05). By the 8-cell stage, NT produced with LD had lower DM than 8- cell HD NT (p<0.05), but was still higher than 8-cell IVP (p<0.05). NT blastocysts produced with LD had DM similar to IVP (p>0.05), and both were lower than HD NT blastocysts (p<0.05). These data indicate that cell lines selected for reduced Dnmt1 expression can reduce global DM in the developing NT embryo. However, DM was not reduced to the levels found in IVP embryos at either the 2- or the 8-cell stages. Future efforts will need to determine if these reductions are adequate and allow for normal development in vivo, or if additional efforts will need to be made to further mimic normal embryonic reprogramming events. This project was supported by National Research Initiative Competitive Grant no. 2006- 35203-16620 from the USDA Cooperative State Research, Education, and Extension Service. (platform) (platform)

DIFFERENTIAL GENE EXPRESSION PROFILING OF DAY 45 PLACENTA IN CHROMATIN TRANSFER AND IN VITRO FERTILIZATION-DERIVED CATTLE FETUSES EVALUATED BY TWO MICROARRAY PLATFORMS

Nuclear transfer has been used to produce genetically identical farm animals for a variety of purposes. Recently, an alternative cloning method called chromatin transfer (CT) was developed to produce cloned cattle. While the success rate of CT is higher than for nuclear transfer it is still far from optimal and is also likely caused by abnormal expression of specific genes. A common abnormality seen in somatic cell-derived pregnancies is Large Offspring Syndrome (LOS). This syndrome involves developmental abnormalities not only in the fetus but also in the placenta. The present study was designed to compare differences in patterns of gene expression between placentas obtained from CT and IVF fetuses using two different microarray platforms. We hypothesized that the abnormal placentation process associated with LOS is due to aberrant gene expression patterns. Two different microarray technologies were used.. Complementary DNA (cDNA) was reverse-transcribed from total RNA of day 45 placentas from two CT and two IVF bovine fetuses and hybridized to two different microarrays. The first was the Pyxis microarray (Pyxis Genomics) which was generated from normalized bovine spleen and placental cDNA libraries containing nearly 7600 double-spotted genes and the second was the Affymetrix GeneChip oligonucleotide array containing more than 23,000 bovine transcripts. The hybridization intensities were Log2-transformed and analyzed by an unpaired t-Test parametric model. Changes in gene expression induced by the treatment (CT) were considered significant if a gene had a p-value < 0.05 and a ±1.1 fold change compared to IVF. A total of 769 genes showed differential gene expression profiles on the affymetrix chip, while the Pyxis array had 233 differentially expressed genes. Cross comparison between the two arrays identified 50 genes showing the same change in expression pattern on the two arrays. These included 11 ESTs, 8 enzymes, 7 adhesion molecules, 5 oncogenes, 12 cell growth and proliferation proteins, 5 cell trafficking molecules, 5 signaling transduction molecules and 5 transcription factors. Quantitative real time PCR (qRT-PCR) was used to validate genes significantly up or down regulated on the microarray platforms. The criteria used to select the genes from the microarrays were based on biological and statistical significance. Out of 10 genes tested, TIMP-3 and TKDP4 were confirmed as up-regulated in the CT samples while p57 and LAMA3 were confirmed to be down-regulated by qRT-PCR. An additional 4 IVF samples and 4 CT samples were analyzed on the Affymetrix array and the same 4 genes were confirmed to be different in these samples. Despite the relatively low number of samples analyzed on the microarrays we were able to identify genes that showed consistent differences in pattern of expression on both arrays. There was a significant number of genes that did not show the same trends on the two microarrays. Future studies will focus on determining how altered expression of these four genes may contribute to the abnormal placentation observed in LOS. (platform)

Comparison of Akabane virus isolated from sentinel cattle in Japan

Adult cows, ewes, and goats infected with Akabane virus (AKAV) of the genus Orthobunyavirus of the family Bunyaviridae do not present any clinical signs; however, in utero infections may result in abortion, premature birth, stillbirth, and congenital deformities such as arthrogryposis-hydranencephaly syndrome in cattle, sheep, and goats. In contrast, the Iriki strain, a variant of AKAV isolated from a calf with nervous signs and encephalitis, causes encephalitis in experimentally inoculated calves. Two AKAV field isolates, named Okayama2001 and Okayama2004, were isolated from blood specimens of sentinel calves and characterized by cross-neutralization testing, genetic analyses of the S and M RNA segments, and experimental intraperitoneal infection in mice. Although a genetic relationship was established between Okayama2001 and the Iriki strain, their antigenic characteristics differ. Okayama2001 was avirulent in mice, as was the OBE-1 strain, which was isolated from an aborted bovine fetus. In contrast, Okayama2004 was antigenically and genetically related to the OBE-1 strain, but was virulent in mice, similar to the Iriki strain. These results indicate that the isolates mutated antigenically or pathogenically and suggest that AKAV mutates frequently in the field. Although attenuated and inactivated vaccines have been developed for disease prevention, an outbreak may occur due to variant viruses arising from mutation.

Ultrasonic transit-time measurement of blood flow in the umbilical arteries and veins in the bovine fetus during stage II of labor

The vitality of the bovine fetus during parturition depends on an intact umbilical circulation to supply adequate amounts of oxygen and nutrients to the fetus. The goal of the present study was to measure the blood flow in the umbilical vessels during stage II of labor and to determine when blood flow ceases in the umbilical cord. In 20 cows, ultrasonographic transducers were placed on one umbilical vein and one umbilical artery after rupture of the allantochorionic sac, and the blood flow volume per unit time was measured. At the same time, a pressure transducer was placed into the uterus to measure uterine pressure. Parturition was spontaneous in all 20 cows. In 20 live calves born, pH, base excess and lactate concentration were measured in the blood immediately after birth. During the last 90 min before birth the mean total umbilical blood flow (artery and vein combined) was 1.186 ± 0.028 L/min. Calves with a blood pH ≥ 7.2 ( n = 13) had a higher mean total blood flow than calves with a pH < 7.2 ( n = 7; 1.243 ± 0.038 versus 1.095 ± 0.038 L/min). In calves with a blood pH < 7.2, the mean total blood flow decreased from 1.178 ± 0.134 at 20 min before birth to 0.959 ± 0.126 L/min at the end of stage II of labor. During this time period, the arterial blood flow did not differ between calves with a blood pH ≥ 7.2 and < 7.2, but venous blood flow decreased significantly in calves with a blood pH < 7.2. During uterine contractions, the total umbilical blood flow decreased significantly by 0.22 L/min. The blood flow in the umbilical artery and vein ceased before the calves were completely born.

Evaluation of bovine abortion associated with Neospora caninum by different diagnostic techniques in Mashhad, Iran

Twelve aborted foetuses (gestational ranged from 4-9 months) and dams from dairies cattle farms in (Mashhad) Iran were analysed to investigate the participation of Neospora caninum in abortion. Diagnosis of the infection was determined by histopathology, serology (indirect fluorescent antibody test [IFAT]) and semi-nested polymerase chain reaction (PCR). A total 33% of bovine foetuses were considered to be infected by PCR technique. Microscopic lesions consistent with N. caninum infection in foetal brains were observed in 25% of the samples, whereas 33% were positive using IFAT (with a cut-off titre of 1:20). This study confirms the importance of N. caninum as an important cause of abortion in Iran.

187 IMPRINTED GENE EXPRESSION IN IN VIVO-AND IN VITRO-PRODUCED BOVINE FETUSES AND PLACENTAS

Some gestational alterations associated with bovine somatic cell nuclear transfer (SCNT) are presumably consequences of abnormal imprinted gene expression. This work aimed to evaluate the expression patterns of imprinted genes IGF2 and IGF2R in bovine fetuses and chorioallantoic membranes derived from in vivo- and in vitro-produced embryos. Fetuses were produced by AI (in vivo group, n = 3), IVF (n = 3), parthenogenesis (n = 3), or SCNT (n = 2). Cows with positive pregnancy diagnosis after ultrasonographic examination were slaughtered between Days 33 and 36 of gestation. The reproductive tract was transported on ice to the laboratory, where fetuses and chorioallantoic fragments were collected and stored in liquid nitrogen. Total RNA extraction was performed using TRIzol, according to manufacturer's instructions, and the reverse transcription reaction was carried out with 1 µg of total RNA, 6.75 µm oligo pd(T)12–18, and 50 U of reverse transcriptase (Improm-II, Promega, Madison, WI, USA). The relative quantification of IGF2 and IGF2R transcripts was done using real-time PCR with SYBR Green dye. The average efficiency of PCR amplifications was estimated for each gene using a linear regression on the logarithm of fluorescence per cycle (Ramakers et al. 2003 Neurosci. Lett. 339, 62–66), and the expression ratios were calculated according to the method described previously by Livak and Schmittgen (2001 Methods 25, 402–408). To verify statistical differences, a pair-wise fixed reallocation randomization test (Pfaffl et al. 2002 Nucl. Acids Res. 30, e36) was used. All expression ratios were normalized by glyceraldehyde 3-phosphate dehydrogenase expression and calibrated by the in vivo group (expression assumed as 1.00 for all genes and tissues). The analysis of relative differences on transcript levels of imprinted genes in fetuses revealed IGF2 down-regulation (P &lt; 0.05) in the SCNT (0.19) and parthenogenetic (0.02) groups when compared to the in vivo group and IVF fetuses (2.02). In chorioallantois, IGF2 was down-regulated (P &lt; 0.001) in parthenotes (0.001) when compared to the in vivo, IVF (3.13), and SCNT (0.98) groups. IGF2R was down-regulated (P &lt; 0.001) in SCNT chorioallantois (0.25) when compared to the in vivo group. Low expression of IGF2 in parthenogenetic fetuses and chorioallantois confirms its imprinted status in bovine. Alterations in the relative frequency of IGF2 and IGF2R transcripts were observed in bovine SCNT-derived fetuses and chorioallantoic membranes, respectively, supporting the hypothesis that abnormalities in the expression of imprinted genes are causes for the low efficiency of SCNT procedures in this species. Such alterations suggest modifications in DNA methylation patterns at IGF2 and IGF2R imprinting centers.

201 LIVER AND KIDNEY FUNCTION IN BOVINE FETUSES FROM EMBRYOS PRODUCED IN VIVO OR IN VITRO

Abnormal offspring syndrome can occur in fetuses and calves resulting from embryos produced in vitro or by nuclear transfer procedures. This study was conducted to determine the effects of in vitro embryo culture on fetal biochemistry profiles and histology of the liver and kidneys during late gestation. Embryos were produced in vivo by using superovulated cows (In Vivo) or in vitro by using a serum-containing culture system (In Vitro) as previously described (Miles et al. 2004 Biol. Reprod. 71, 1919–1926). Single blastocysts from each embryo production system were transferred nonsurgically into heifers. On Day 222 of gestation, fetuses from the In Vivo group (n = 12) and the In Vitro group (n = 12) were recovered in utero. Samples of fetal serum were collected for biochemical analysis. Samples of liver and kidney were prepared for histological evaluation. Stereological methods were used to determine the volume density of hepatocytes as well as kidney glomeruli and kidney tubules. Fetuses from the In Vitro group were heavier (P = 0.03) than those from the InVivo group (17.3 � 1.0 kg and 20.7 � 1.0 kg for InVivo and InVitro, respectively; least squares means � SEM). Liver and paired kidney weights per kilogram of body weight did not differ (P ≥ 0.10) with treatment (26.4 � 0.6 g kg–1 v. 27.6 � 0.6 g kg–1 and 7.8 � 0.5 g kg–1 v. 9.1 � 0.5 g kg–1 for liver and kidney, respectively). In addition, there was no effect of treatment on the volume densities of hepatocytes, kidney glomeruli, and kidney tubules. However, compared with the In Vivo group, fetuses from the In Vitro group had increased (P ≤ 0.02) concentrations of blood urea nitrogen (BUN; 13.8 � 1.8 mg dL–1 v. 19.8 � 1.8 mg dL–1) and BUN:creatinine ratio (4.6 � 0.8 v. 7.9 � 0.8). No differences were observed between the In Vivo and In Vitro groups for serum levels of creatinine, total bilirubin, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, glucose, insulin, and insulin-like growth factor-I. In summary, compared with bovine fetuses from in vivo-produced embryos, fetuses from in vitro-produced embryos had increased body weight, normal liver and kidney morphology, and increased concentrations of BUN during late gestation. Supported by the State of North Carolina.

2 DETECTION OF ANTISENSE TO Igf2r (AIR) RNA IN CATTLE

The insulin-like growth factor type 2 receptor (Igf2r) regulates fetal growth by removing Igf2 from circulation, thus preventing overgrowth. In mice, expression of the Igf2r gene is imprinted only after implantation and is associated with expression of the antisense non-coding (nc)RNA, Air. In contrast, the human IGF2R gene is not imprinted and AIR ncRNA does not exist. Because it is known that the Igf2r gene is imprinted in cattle, the objectives of this study were to determine if Air ncRNA exists in cattle and, if so, whether bovine Air (bAir) is expressed during both pre- and post-implantation development. For objective 1, primer sets were designed for bAir based on bovine genomic sequence. The primer set, bAir3, was used to amplify a region of bAir corresponding to an antisense segment within intron 1 of Igf2r. Primer set bAir4 amplified a segment of bAir ncRNA corresponding to an antisense region upstream of the 52-untranslated region of Igf2r. Pools of whole-cell RNA were extracted from bovine fetal liver and subjected to DNase treatment, reverse transcription (RT), and PCR. Control RT reactions included RT without superscript and RT without superscript or DNase. Controls confirmed that amplification products resulted from RNA present in the sample and not from genomic DNA contamination. Amplicons were obtained for both the bAir3 and the bAir4 primer sets and were sequence verified, demonstrating that bAir ncRNA does exist in cattle. For objective 2, conceptuses (n = 4; mean � SEM length: 2.8 � 0.3 mm) derived from transfer of frozen-thawed in vivo-produced blastocysts were recovered from cows on Day 15 of gestation and snap-frozen for RNA extraction. Samples of liver from in vivo-produced bovine fetuses recovered at Day 70 of gestation (n = 7) were snap-frozen for RNA extraction. Semi-quantitative RT-PCR assays were performed to assess levels of mRNA for Igf2r and H2a, as well as ncRNA for bAir. All conceptus and fetal liver cDNA samples were run in duplicate within the same assay. Relative RNA expression was calculated as the ratio of band intensities of the RNA of interest to that of H2a. Data for relative RNA expression were analyzed by Student's t-test. H2a and Igf2r mRNAs were expressed in all Day 70 fetal liver and Day 15 conceptus samples. Relative levels of Igf2r did not differ (P = 0.19) with stage of development (0.15 � 0.09 v. 0.36 � 0.12 for Day 70 v. Day 15). bAir ncRNA was expressed in 7 of 7 samples of Day 70 fetal liver, whereas only 1 of 4 conceptuses expressed a faint bAir ncRNA signal based on either the bAir3 or bAir4 primer sets (χ2 = 7.23, P &lt; 0.01). Relative levels of bAir ncRNA were greater (P &lt; 0.001) in Day 70 fetal liver compared to those in Day 15 conceptuses for amplicons bAir3 (0.376 � 0.039 v. 0.028 � 0.051) and bAir4 (0.101 � 0.008 v. 0.003 � 0.010). In conclusion, the antisense ncRNA, Air, does exist in cattle and its relative expression is greatest following implantation. These observations are consistent with murine data and suggest that bAir may be involved in regulating imprinted expression of Igf2r in cattle.

Target genes of myostatin loss-of-function in muscles of late bovine fetuses

BackgroundMyostatin, a muscle-specific member of the Transforming Growth Factor beta family, negatively regulates muscle development. Double-muscled (DM) cattle have a loss-of-function mutation in their myostatin gene responsible for the hypermuscular phenotype. Thus, these animals are a good model for understanding the mechanisms underpinning muscular hypertrophy. In order to identify individual genes or networks that may be myostatin targets, we looked for genes that were differentially expressed between DM and normal (NM) animals (n = 3 per group) in the semitendinosus muscle (hypertrophied in DM animals) at 260 days of fetal development (when the biochemical differentiation of muscle is intensive). A heterologous microarray (human and murine oligonucleotide sequences) of around 6,000 genes expressed in muscle was used.ResultsMany genes were found to be differentially expressed according to genetic type (some with a more than 5-fold change), and according to the presence of one or two functional myostatin allele(s). They belonged to various functional categories. The genes down-regulated in DM fetuses were mainly those encoding extracellular matrix proteins, slow contractile proteins and ribosomal proteins. The genes up-regulated in DM fetuses were mainly involved in the regulation of transcription, cell cycle/apoptosis, translation or DNA metabolism. These data highlight features indicating that DM muscle is shifted towards a more glycolytic metabolism, and has an altered extracellular matrix composition (e.g. down-regulation of COL1A1 and COL1A2, and up-regulation of COL4A2) and decreased adipocyte differentiation (down-regulation of C1QTNF3). The altered gene expression in the three major muscle compartments (fibers, connective tissue and intramuscular adipose tissue) is consistent with the well-known characteristics of DM cattle. In addition, novel potential targets of the myostatin gene were identified (MB, PLN, troponins, ZFHX1B).ConclusionThus, the myostatin loss-of-function mutation affected several physiological processes involved in the development and determination of the functional characteristics of muscle tissue.

Detecção de ácidos nucléicos de Brucella spp., Leptospira spp., herpesvirus bovino e vírus da diarréia viral bovina, em fetos bovinos abortados e em animais mortos no perinatal

Samples of 114 bovine fetuses and 10 calves, which dead in perinatal period, were examined for detection of DNA. The most common detected agent was Brucella spp. in 17 samples (13.7%) followed by Leptospira spp. in 4 cases (3.2%),bovine herpesvirus (BHV) and bovine viral diarrhea (BVDV) in 3 animals (2.4%) each, and 1 for the association of BVDV and BHV. In 77.4 % (96/124) of the samples it was not possible to detect any agent.

First report of Neospora caninum-associated bovine abortion in Mashhad area, Iran

Neospora caninum is an apicomplexan protozoan which causes abortion in cattle worldwide. The present study was designed to assess the importance of bovine neosporosis for causing abortion in Iranian cattle. Infection was primarily diagnosed by polymerase chain reaction (PCR), complemented with histopathology and immunohistochemistry (IHC). One hundred brains of aborted bovine fetuses were collected from Iranian dairy herd between 2003 and 2005 in Mashhad area. N. caninum was detected by PCR in the brains of 13 aborted fetuses. Lesions consistent with N. caninum infection were observed in 12 of fetal brains. Immunohistochemical examination of brain detected N. caninum organisms in three aborted fetuses, and a thick-walled (2 mum) cyst with 50 mum diameter was identified in one of the IHC-positive brain. The results indicated that neosporosis is an important cause of abortion in dairy cattle of Iran.

Optimizing Bovine Abortion Diagnostics

Very few aborted bovine fetuses have distinctive pathologic changes that contribute to confirming a diagnosis, with the exception of fetuses with congenital anomalies, thus sending an intact fetus to a diagnostic laboratory for necropsy by a pathologist rarely enhances the prospects for obtaining a positive diagnosis. In fact, it is questionable whether necropsies of aborted fetuses routinely merit the time of a busy veterinary practitioner, when a technician can be trained to collect, package and ship specimens as effectively and more efficiently. In addition, owners, managers, and herdsmen can be recruited into the diagnostic team in herds with on-going abortion problems if proper training and supplies are provided. A protocol for proper specimen collection and packaging for diagnosis of bovine abortions, which can be used by veterinarians or for training technicians, will be presented.

Isolation of Bovine Cardiomyocytes for Reprogramming Studies Based on Nuclear Transfer

The goal of this study was to establish and validate a protocol for preparing bovine cardiomyocytes from slaughterhouse material for nuclear transfer experiments. The cardiomyocyte was selected because it is a terminally differentiated cell and strongly expresses a unique subset of genes which can be monitored during the reprogramming period. A total of 39 trials were conducted, and an optimized protocol was developed yielding individual contractile cardiomyocytes from 3-5-month-old bovine fetuses The basic protocol involves stabilization of bovine heart tissue for transportation from the slaughterhouse to the laboratory by perfusion with Custodiol. This was followed by an enzymatic dissociation with collagenase in calcium-free medium and yielded individual contractile rod-shaped cardiomyocytes. Subsequent addition of Ca2+ caused the cardiomyocytes to round up which was an essential pre-condition for drawing them into glass transfer pipettes for delivery into the perivitelline space and for efficient electrofusion with cytoplasts derived from in vitro matured bovine oocytes. The use of cardiomyocytes maintained at 37 degrees C in nuclear transfer, resulted in a significantly reduced proportion of blastocysts compared to adult fibroblasts (14.0% versus 32.7%). Storage of cardiomyocytes at 4 degrees C prior to nuclear transfer was not compatible with blastocyst development. It is expected that this system will be valuable for investigating the reprogramming of gene expression which occurs after somatic cell nuclear transfer.

Developmental Aberrations of Liver Gene Expression in Bovine Fetuses Derived from Somatic Cell Nuclear Transplantation

Cloning by somatic cell nuclear transfer (NT) has been accomplished. However, the process itself is inefficient since most clones die before birth and survivors often display various anomalies. In an effort to determine global expression profiles of developmentally regulated liver genes in NT bovine fetuses, we employed a custom-made bovine liver complementary DNA (cDNA) microarray. The NT fetuses in early pregnancy were derived from cumulus cells as the nuclear donor cells. Normal fetuses were derived from in vitro fertilization (IVF) and artificial insemination (AI). Gene expression levels in NT, IVF, and AI fetal livers were obtained by comparing individual fetal liver samples with that of adult liver of nonpregnant cycling cows. Statistical analyses of the expression data showed widespread dysregulation of developmentally important genes in the three NT fetuses examined. It was found that the number of dysregulated genes was within a range of 3.5-7.7% of the tested genes in the NT fetal livers. The analyses revealed that one NT fetus was markedly different in liver gene expression profile from the other two NT fetal livers in which the expression profiles were highly correlated. Thus, our findings demonstrate that widespread dysregulation of liver genes occurs in the developing liver of NT bovine fetuses. It is possible that inappropriate genomic reprogramming after NT is a key factor associated with abnormal gene expressions in the livers of NT fetuses, whereas distinct expression patterns between the fellow cloned fetuses likely have resulted from variable epigenetic status of the donor nuclei.