Evaluation of porcine circovirus type 2 infection in in vitro embryo production using naturally infected oocytes.

In vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) are important breeding techniques for livestock. High-quality MII oocytes produced from in vitro maturation (IVM) are required for the two techniques listed above. The ovaries used for IVM operations are primarily acquired from commercial abattoirs, and the pathogen status of slaughtered animals becomes an unavoidable issue. Our previous monitoring data showed that porcine circovirus type 2 (PCV-2) is the main pathogen present in ovaries from abattoirs. However, the characteristics and effects of PCV-2 infection in oocyte maturation and in vitro production (IVP) of embryos are unclear, and currently there are no relevant studies. Therefore, the aim of this study was to determine the PCV-2 infection pattern and determine whether it affects oocyte in vitro maturation and IVP embryo development. More than five hundred ovaries and five thousand oocytes were utilized in the present study. Polymerase chain reaction (PCR) was used to detect PCV-2 DNA in ovaries, follicular fluid (FF), oocytes, cumulus cells and IVP embryos. The effects of viral infections on the rate of oocyte maturation and IVP embryo development were evaluated. We also analyzed the number of copies of the virus in the IVM and IVP process by absolute quantitative fluorescence PCR. Our study showed that the prevalent virus subgenotype in ovaries was PCV-2a. PCV-2a infection did not significantly affect ovarian/oocyte morphology and maturation. Moreover, virus infection did not have a significant effect on the development of the IVP embryos except for a reduction in IVF blastocyst cell numbers. Further tests showed that the viral copy numbers fluctuated at different stages between the IVP embryos and culture medium. For the first time, this study identified the infection pattern of naturally sourced PCV-2 in the course of oocyte maturation and embryo development.

Effects of post-partum administration of ketoprofen on sow health and piglet growth.

The effect of the non-steroidal anti-inflammatory drug ketoprofen on the post farrowing phase of sows was studied in a randomized, blinded, placebo-controlled trial. Ketoprofen (3mg/kg) was administered intramuscularly to 20 healthy sows for 3 days post-partum (p.p.). The control group (n=20) received a saline placebo. Backfat, number of days of constipation and days before feed refusal were measured. Body condition (BCS) and shoulder sores were scored for 1 week p.p. Changes in BCS, backfat and shoulder sore scores were analysed with ANOVA. Blood was collected on days -1, 0, 5 and 14 with respect to medication. Aspartate aminotransferase (AST), creatinine kinase (CK), haptoglobin and serum amyloid A (SAA) were quantified and analysed with a Mann-Whitney U test. BCS and backfat decreased less following ketoprofen administration than with the placebo (-0.08 ± 0.2 vs. -0.8 ± 0.2, 1.0 ± 0.8mm vs. -2.0 ± 0.9 mm, respectively; P<0.05 for both) during the first 2 weeks of lactation. The shoulder sore score deterioration was milder during days 4-6 p.p. with ketoprofen than placebo (P<0.05). Duration of constipation was shorter with ketoprofen than placebo (5.5 ± 0.3 vs. 6.4 ± 0.3 days p.p.; P<0.05). Incidences of feed refusal occurred later in the ketoprofen group than in the placebos (9.6 ± 0.9 vs. 3.8 ± 0.8 days p.p.; P<0.05). AST and SAA values were higher after ketoprofen administration than placebo on day 5 p.p. (P<0.05). It was concluded that ketoprofen appeared to benefit sows during the first 2 weeks post farrowing, but caused some tissue irritation.

Aneuploidy detection in pigs using comparative genomic hybridization: from the oocytes to blastocysts.

Data on the frequency of aneuploidy in farm animals are lacking and there is the need for a reliable technique which is capable of detecting all chromosomes simultaneously in a single cell. With the employment of comparative genomic hybridization coupled with the whole genome amplification technique, this study brings new information regarding the aneuploidy of individual chromosomes in pigs. Focus is directed on in vivo porcine blastocysts and late morulas, 4.7% of which were found to carry chromosomal abnormality. Further, ploidy abnormalities were examined using FISH in a sample of porcine embryos. True polyploidy was relatively rare (1.6%), whilst mixoploidy was presented in 46.8% of embryos, however it was restricted to only a small number of cells per embryo. The combined data indicates that aneuploidy is not a prevalent cause of embryo mortality in pigs.

Microarray analysis reveals genes and functional networks relevant to the predisposition to inverted teats in pigs.

The inverted teat defect is characterized by the failure of teats to protrude from the udder surface and has a negative effect on the economic efficiency of pig production. The inverted teat defect is influenced by genetic factors, but the number and identity of relevant genes are unknown. In this study, we compared the mRNA expression of teat tissues from unaffected pigs and affected pigs by using microarrays. Simultaneously, 24,123 probe sets were screened, of which some 15,000 had present calls and were analyzed for differential expression between mesenchymal and epithelial tissue of 3 categories of teats (i.e., normal teats of unaffected and affected animals, and inverted teats of the latter). Differential expression was more pronounced in epithelial than in mesenchymal tissue, and the comparisons among the 3 categories of teats showed that local processes at the side of the affected area as well as processes taking place at the level of the organ contribute to the development of inverted teats. Genes related to biofunctions of cell maintenance, proliferation, differentiation, and replacement; organismal, organ, and tissue development; genetic information and nucleic acid processing; and cell-to-cell signaling and interaction were differentially expressed, depending on the teat phenotype and the status of the animal as affected or unaffected. In particular, genes encoding members of canonical pathways of growth factor signaling were highlighted. Complementary to previous real-time quantitative reverse-transcription PCR experiments showing upregulation of growth factors (epidermal growth factor, fibroblast growth factor, hepatocyte growth factor, platelet-derived growth factor, vascular endothelial growth factor) and their receptors in the inverted teat, here it is shown that the abundance of transcripts encoding subordinated proteins (acid phosphatase 1, soluble; activating transcription factor 2; casein kinase 2, α 1 polypeptide; casein kinase 2, α prime polypeptide; actinin, α 2; and Homo sapiens growth factor receptor-bound protein 2) within the growth factor signaling pathways are also affected. Tuning of the expression of genes of these pathways balances the differentiation and proliferation of epithelial and mesenchymal teat tissue and finally affects the shape and structure of the teats.

Outcome of experimental porcine circovirus type 1 infections in mid-gestational porcine foetuses.

BACKGROUND: Porcine circovirus type 1 (PCV1) has been described as a non-cytopathic contaminant of the PK-15 cell line. Several experimental infections with PCV1 failed to reproduce disease in pigs. Therefore, PCV1 is generally accepted as non-pathogenic to pigs. To our knowledge, nothing is known about the outcome of PCV1 infections in porcine foetuses. This was examined in the present study. RESULTS: Nine foetuses from three sows were inoculated at 55 days of gestation: three with 10(4.3) TCID(50) of the PCV1 cell culture strain ATCC-CCL33, three with 10(4.3) TCID(50) of the PCV1 field strain 3384 and three with cell culture medium (mock-inoculated). At 21 days post-inoculation, all 6 PCV1-inoculated and all 3 mock-inoculated foetuses had a normal external appearance. Microscopic lesions characterized by severe haemorrhages were observed in the lungs of two foetuses inoculated with CCL33. High PCV1 titres (up to 10(4.7) TCID(50)/g tissue) were found in the lungs of the CCL33-inoculated foetuses. All other organs of the CCL33-inoculated foetuses and all the organs of the 3384-inoculated foetuses were negative (< 10(1.7) TCID(50)/g tissue) by virus titration. PCV1-positive cells (up to 121 cells/10 mm(2) in CCL33-inoculated foetuses and up to 13 cells/10 mm(2) in 3384-inoculated foetuses) were found in the heart, lungs, spleen, liver, thymus and tonsils. PCR and DNA sequencing of Rep recovered CCL33 or 3384 sequences from CCL33- or 3384-inoculated foetuses, respectively. CONCLUSIONS: From this study, it can be concluded that cell culture PCV1 can replicate efficiently and produce pathology in the lungs of porcine foetuses inoculated at 55 days of foetal life.

Real-time PCR to detect and analyze virulent PPV loads in artificially challenged sows and their fetuses.

To establish a real-time polymerase chain reaction with SYBR Green for detection and quantification of porcine parvovirus (PPV) in porcine tissues, two primers specific for the non-structural protein 1 gene were designed. The detection limit of this assay was 3-23 gene copies/reaction, equivalent to 0.001 TCID(50)/ml. The assay was linear over a 10(6) dilution range of template concentrations. Other porcine pathogens involved in reproductive disorders (porcine circovirus 2, porcine reproductive and respiratory virus, pseudorabies virus, classical swine fever virus) were negative by this assay. This assay could detect PPV titres at least 10(5) smaller than the hemagglutination assay. To better understand the pathogenesis of PPV, the levels of viral DNA in various tissues of artificially challenged sows and their fetuses were quantified with this method. The virus was found mainly in the heart, lung, spleen, kidney, and endometrium of sows, and mainly in the heart, spleen, lung, and testis of fetuses. This study provides a new tool for the study of PPV infection and distribution in sows and their fetuses.

The blastocyst production rate and incidence of chromosomal abnormalities by developmental stage in in vitro produced porcine embryos.

The present study was conducted to determine the relationship between embryonic development speed at different stages (the cleaved stage at 52 h and the blastocyst stage at 6 days post insemination) and incidences of chromosome abnormalities in in vitro produced porcine embryos. Porcine oocytes were collected from 3-6-mm ovarian follicles obtained at a slaughterhouse and matured in modified NCSU-37 medium for 44-46 h. Following in vitro fertilization with a final concentration of 1 x 10(5) sperm/ml for 3 h, all oocytes were cultured in vitro for 52 h. Day-2 (52 h after insemination) embryos were classified according to their cleaved stages into 2-cell, 3- to 4-cell, 5- to 8-cell, and >8-cell stages; these were cultured separately for additional 4 days (Day 6). The resultant Day-6 blastocysts were classified according to the morphological diameter into 3 grades: Grade A, expanded blastocysts; Grade B, expanding blastocysts; and Grade C, early blastocysts. They were then analyzed chromosomally. The 3- to 4-cell and 5- to 8-cell embryos had significantly high blastocyst development rates (46.1 and 36.9%, respectively), and these blastocysts contained significantly more cells (40.2 and 42.4 cells, respectively) than those derived from 2-cell embryos and >8-cell embryos (28.6 and 26.5 cells, respectively). The incidence of chromosomal abnormalities was significantly higher in the blastocysts derived from 2-cell and >8-cell stage embryos than in the blastocysts derived from the other stage embryos. Furthermore, the grade A blastocysts had the lowest incidence of chromosomal abnormalities (35.3%) and contained the most cells (48.7 cells). Porcine in vitro production (IVP) yielded a high blastocyst rate and an excellent embryo quality when 3- to 4-cell and 5- to 8-cell stage embryos were selected on Day 2 after insemination. The same criteria yielded a higher quality of expanded blastocysts based on the stage of embryo development and morphology.

Impacts on conceptus survival in a commercial swine herd.

An estimated 30 to 40% of potential piglets are lost before farrowing in U.S. or European pig breeds. Because these studies were conducted in limited numbers of university research herds, we decided to characterize the timing, pattern, and extent of conceptus loss in a commercial swine herd in Iowa (Pig Improvement Company; Camborough Line). Sows (parities 2 to 14) were slaughtered on d 25 (n = 83), 36 (n = 78), or 44 (n = 83) of gestation. These days coincide with periods before, during, and after uterine capacity becomes limiting to conceptus survival. At slaughter, numbers of corpora lutea (CL) and uterine horn length were determined, and conceptuses were removed and evaluated. Uterine horn length and CL number did not differ among these days of gestation, averaging 217 cm and 26.6 CL, respectively. In contrast, numbers of conceptuses decreased (P < 0.05) from 15.8 on d 25 to 12.9 on d 36, then remained relatively constant through d 44 (12.1). Thus, conceptus survival averaged 60.2% on d 25, 50.1% on d 36, and 46.3% on d 44, based on numbers of CL present. There was a positive correlation (P < 0.001; r = 0.50) between numbers of viable conceptuses on d 25 and ovulation rate, but this association was completely lost by d 36 (P > 0.10) when uterine capacity becomes limiting. In agreement with this premise, uterine horn length and conceptus number were not associated on d 25 but exhibited positive correlations (P < 0.05) on d 36 (r = 0.36) and d 44 (r = 0.40). On all 3 d examined, the numbers of viable conceptuses were not associated with fetal weight but were negatively correlated (P < 0.05) with placental weight. Compared with the commonly reported values for ovulation rate and percentage conceptus loss in university research herds, values from these production animals were extremely high. Data suggest that throughout this period, larger litters were associated with conceptuses exhibiting small placentae. These data lend support to the concept that increased placental efficiency (fetal weight/placental weight) may contribute to increased litter size in the pig.

Antibody repertoire development in fetal and neonatal piglets. IV. Switch recombination, primarily in fetal thymus, occurs independent of environmental antigen and is only weakly associated with repertoire diversification.

The epitheliochorial placenta of swine is considered a barrier to Ag and selective transport of IgG, so this species should be an excellent model with which to determine whether switch recombination is Ag dependent. Analysis of Ig levels and Ig isotype profiles in >150 normal and virus-infected fetuses from 38-110 days of gestation (DG) suggested that IgG, IgA, and IgM were most likely the result of de novo fetal synthesis. Although transcripts for IgM could be recovered at DG 50 (114 DG is full gestation) in all major fetal lymphoid tissues, those for IgG and IgA first became prominent at 60 DG in thymus, and transcription and spontaneous secretion became especially pronounced in this organ in older fetuses. Data on transcription, secretion, and serum isotype profiles suggest that although all fetal IgA and IgM may result from de novo synthesis, some IgG may result from low-level selective transport. The complementarity-determining region 3 spectratypes of thymic IgA and IgG transcripts at 70 and 90 days, respectively, were as polyclonal as that of IgM, indicating a broad repertoire of switched B cells although the VDJs transcribed with these switched isotypes in normal fetuses were not diversified in comparison to those from animals exposed to environmental Ags such as age-matched, virus-infected fetuses, colonized isolator piglets, and conventional adults. However, VDJs expressed with switched isotypes were more diversified than those expressed with IgM. Thus, switch recombination in fetal life does not appear to be driven by environmental Ag and is only weakly coupled to VDJ diversification. These findings, and the fact that the oligoclonal IgA and IgM repertoires in a noninductive site of the mucosal immune system (parotid gland) become polyclonal in piglets reared germfree, suggest that initial expansion of the switched cells in the B cell compartment of fetal and neonatal piglets is not driven by environmental Ag.

Porcine circovirus 2 infection in swine foetuses inoculated at different stages of gestation.

The ability of porcine circovirus 2 (PCV2) to replicate and cause pathologic abnormalities in foetuses at selected time points of gestation was examined in this study. Two foetuses were inoculated in utero in each of two sows at 57, 75 and 92 days of gestation, respectively, with PCV2 (1121). The remaining foetuses were left uninoculated to assess whether intra-uterine spread occurred. Twenty-one days after inoculation, the foetuses were collected and examined for gross lesions and for virus and infected cells in different organs. Serum samples from all foetuses were tested for PCV2 antibodies. Virus replication was detected in all inoculated foetuses. Spread to non-inoculated foetuses did not occur. Virus replication was significantly higher in foetuses inoculated at 57 days compared to that inoculated at 75 and 92 days. The heart contained the highest virus titre and highest number of viral antigen positive cells. Gross lesions were observed only in foetuses inoculated at 57 days of age. PCV2 antibodies were detected only in foetuses inoculated at 75 and 92 days. This study shows the ability of PCV2 to replicate in foetuses at different stages of gestation and to cause pathologic abnormalities in foetuses inoculated at 57 gestational days.

Maternal dietary protein deficiency decreases amino acid concentrations in fetal plasma and allantoic fluid of pigs.

This study was conducted to test the hypothesis that maternal dietary protein deficiency decreases amino acid availability to the fetus, thereby contributing to retarded fetal growth. Primiparous gilts selected genetically for low or high plasma total cholesterol concentrations (low line and high line, respectively) were mated, and then fed 1.8 kg/d of isocaloric diets containing 13% or 0.5% crude protein. At d 40 or 60 of gestation, they were hysterectomized, and maternal and fetal blood samples as well as amniotic and allantoic fluids were obtained for analyses of amino acids, ammonia and urea. Dietary protein restriction decreased (P < 0.05) the following: 1) maternal plasma concentrations of urea at d 40 and 60 of gestation; 2) fetal plasma concentrations of alanine, arginine, branched-chain amino acids (BCAA), glutamine, glycine, lysine, ornithine, proline, taurine, threonine and urea at d 60 of gestation; 3) amniotic and allantoic fluid concentrations of urea at d 40 and 60 of gestation; and 4) allantoic fluid concentrations of alanine, arginine, BCAA, citrulline, cystine, glycine, histidine, methionine, proline, serine, taurine, threonine and tyrosine at d 40 of gestation, in gilts of both genetic lines. At d 60 of gestation, protein deficiency decreased (P < 0.05) allantoic fluid concentrations of arginine, cystine, glycine, taurine and tyrosine in low line gilts and of cystine, glutamine, ornithine, serine, taurine and tyrosine in high line gilts. Low line and high line gilts also differed remarkably in allantoic fluid concentrations of arginine, glutamine, ornithine and ammonia at d 40 and 60 of gestation. Our results suggest the following: 1) protein-deficient gilts maintain maternal plasma concentrations of amino acids by mobilizing maternal protein stores and decreasing oxidation of amino acids during the first half of gestation; 2) protein deficiency may impair placental transport of amino acids from the maternal to the fetal blood; and 3) low line and high line gilts differ in fetal amino acid metabolism. Decreases in concentrations of the essential and nonessential amino acids in the fetus may be a mechanism whereby maternal dietary protein restriction results in fetal growth retardation.

The onset of maternal diabetes in swine induces alterations in the development of the fetal preadipocyte.

Diabetes induced during gestation has previously been demonstrated to increase adipose accretion in the fetal pig. The present experiment examined whether maternal diabetes alters the proliferation and differentiation of the fetal preadipocyte. Seven crossbred gilts were injected with alloxan (50 mg/kg) at d 75 of gestation to induce diabetes and seven additional gilts were injected with buffer (controls). All gilts underwent Caesarean section of d 105 of gestation. Cells obtained from adipose tissue of fetuses of diabetic swine (FDS) at d 105 of gestation demonstrated a greater (P < .05) proliferative response (57%) and higher (P < .05) rates of differentiation as determined by sn-glycerol-3-phosphate dehydrogenase (142% increase) and lipoprotein lipase (80% increase) activities than cells acquired from fetuses of control swine (FCS). Adipogenic activity of the sera from these two groups of fetuses did not differ when tested on adipose tissue from fetuses at 105 d of gestation. However, use of these sera on cells derived from normal fetuses at 75 d of gestation resulted in detection of an increase (P < .05) in adipogenic activity within the sera from FDS. This study suggests that maternal diabetes causes alterations in the preadipocyte fraction of cells within the developing adipose tissue that result in formation of more adipocytes and thus permits greater capacity for lipid accumulation in the growing fetus of the diabetic pig. These alterations in the preadipocyte result from the activity of factors that transitionally function during the latter half of gestation.

Ectopia cordis thoracoabdominalis in a piglet.

A congenital anomaly characterised by displacement of the heart through a ventral body wall fissure involving the thoracic and cranial abdominal regions was recorded in a female Yorkshire-cross piglet. Dissection to assess the morphology of the developmental defect and a summary review of the literature on ectopia cordis were made. This case appears to be one of only three of ectopia cordis thoracoabdominalis reported in swine.

Semitendinosus muscle development in fetally decapitated pigs.

The effect of decapitation on semitendinosus muscle growth and development was studied in the fetal pig. Pig fetuses were decapitated at 45 d of gestation and histochemical and biochemical analyses conducted at 110 d of gestation. Decapitation prevented or delayed the conversion of secondary fibers from Type II to Type I histochemistry as is characteristic of the normal semitendinosus. Morphological analyses indicated either a prolonged stage of secondary myofiber formation or an inhibition of secondary myofiber hypertrophy in muscle from decapitated fetuses. Biochemical and histochemical analyses indicated that muscle from decapitated fetuses was biochemically and chemically immature compared with that from normal fetuses.

Fertility, hatchability and malformations in guinea fowl embryos as affected by dietary manganese.

1. The effects of feeding diets containing 54 mg, 60 mg, 65 mg or 70 mg manganese/kg to Guinea fowls on fertility, hatchability and the occurrence of malformed embryos were studied. 2. Dietary manganese affected fertility slightly, but significantly affected hatchability and the occurrence of malformed embryos. 3. Hatchability was most significantly depressed at the lowest dietary concentration of manganese, which also caused a highly significant increase in the proportion of malformed embryos. 4. Although increasing manganese to more than 54 mg/kg improved hatchability and reduced embryonic malformations, the increase did not completely eliminate the latter condition.

The pathogenesis of congenital hereditary lymphedema in the pig.

The morphogenesis of congenital hereditary lymphedema was studied in pigs. The disorder, which is essentially a general underdevelopment or even total non-development of the lymphatic system proved to be present during the whole period of lymphatic development. It is suggested that a retardation in the differentiation of the lymphatic primordia from the primitive veins is the early event regulated by a chromosomal aberration. The longer this delay the more serious the lymphatic malformations.