Evaluation of the BioFire FilmArray® GastrointestinalPanel in a Midwestern Academic Hospital.

The BioFire FilmArray® Gastrointestinal Panel (GIP) was implemented to replace traditional stool culture and enzyme immunoassay (EIA) testing for stool pathogens. The purpose of this study was to evaluate the detection rate, incidence of coinfection, and culture recovery rate of gastrointestinal (GI) pathogens detected by the GIP over a 1-year period. A total of 2257 stools collected from January to December 2015 were tested using the GIP. Clostridium difficile colonization was also evaluated by an antigen/toxin EIA and confirmatory polymerase chain reaction (PCR). The GIP detected one pathogen in 911 (40.4%) specimens. Coinfections were detected in 176 (7.8%) of these specimens. The most frequently detected pathogens were C. difficile (15.2%), norovirus (8.9%), enteropathogenic Escherichia coli (7.1%), enteroaggregative E. coli (3.4%), Campylobacter spp. (2.3%), and sapovirus (2.0%). Each of the remaining GIP targets had a detection rate of ≤1.6%. The recovery of bacteria for public health investigations varied, with rates as high as 77% for Salmonella to as low as 30% for Yersinia enterocolitica. Of stools positive for C. difficile on the GIP that were tested by EIA, only 42.7% (88/206) were found to be producing detectable toxin. Overall, the implementation of the GIP resulted in high detection rates of GI pathogens, including the frequent detection of coinfections. This is a promising test to streamline the testing of agents causing infectious gastroenteritis from multiple tests down to a single order with limited hands-on time. Ongoing studies will need to assess the impact that the GIP has on downstream patient care and public health practices.

Assessment of an AI-based tool for population-wide collection of placental morphological data.

OBJECTIVES: Automated placental assessment could allow accurate and timely morphological/pathological measurements at scale. We undertook a pilot study using an artificial intelligence-based assessment system (AI-PLAX) to ascertain the potential of a state-wide rollout as part of Generation Victoria, assessing the impact of time post-delivery, user, and technology used for image capture, on a range of derived placental data. STUDY DESIGN: Ten placentas were imaged by three different users and imaging technologies (iPad, iPhone, Samsung) at (0 h), 24 h, and 48 h post-delivery. Using AI-PLAX, disc size (short and long length, perimeter, area), shape (normal, abnormal), cord insertion type (central, eccentric), cord coiling, abruption (retroplacental hematoma), and meconium staining were determined. RESULTS: When analysing the maternal surface of the placenta, time in cold storage post-delivery had modest effects on placental dimensions, with decreases in the short length (24-48 h: -3.7 %), disc area (0-24 h: 4.7 % and 0-48 h: -7.4 %), and perimeter (0-48 h: -3.8 %) observed. There was marginal impact on placental dimensions when the placenta was imaged by different users, including long length (+1.9 %), disc area (+2.9 %), and perimeter (+2.0 %). Measures of placental size were not impacted by the type of technology used to capture the images. When analysing the fetal surface of the placenta, more variance in placental size measures were observed between users. Abruption detection was not affected by any parameter. Time between delivery and imaging impacted apparent meconium staining - likely reflecting changes in fetal surface colour over time. Meconium staining was not affected by technology or user. CONCLUSIONS: This study supports the feasibility of the collection of placenta images for later morphological analysis by AI-PLAX, with measures obtained minimally influenced by time in cold storage, user imaging the placenta, or technology to capture the images.

Vascular endothelium-specific overexpression of human catalase in cloned pigs.

The objective of this study was to develop transgenic Yucatan minipigs that overexpress human catalase (hCat) in an endothelial-specific manner. Catalase metabolizes hydrogen peroxide (H(2)O(2)), an important regulator of vascular tone that contributes to diseases such as atherosclerosis and preeclampsia. A large animal model to study reduced endothelium-derived H(2)O(2) would therefore generate valuable translational data on vascular regulation in health and disease. Yucatan minipig fetal fibroblasts stably co-transfected with human catalase (Tie2-hCat) and eGFP expression constructs were isolated into single-cell populations. The presence of the Tie2-hCat transgene in individual colonies of fibroblasts was determined by PCR. Transgenic fibroblasts were used for nuclear transfer into enucleated oocytes by electrofusion. A minimum of 140 cloned embryos were transferred per surrogate sow (n = 4). All four surrogates maintained pregnancies and piglets were delivered by cesarean section. Nine male piglets from three of the four litters carried the Tie2-hCat transgene. Expression of human catalase mRNA and overall elevated catalase protein in isolated umbilical endothelial cells from transgenic piglets were verified by RT-PCR and western blot, respectively, and endothelial localization was confirmed by immunohistochemistry. Increased enzymatic activity of catalase in transgenic versus wild-type endothelial cells was inferred based on significantly reduced levels of H(2)O(2) in culture. The similarities in swine and human cardiovascular anatomy and physiology will make this pig model a valuable source of information on the putative role of endothelium-derived H(2)O(2) in vasodilation and in the mechanisms underlying vascular health and disease.

Identification and quantification of differentially represented transcripts in in vitro and in vivo derived preimplantation bovine embryos.

Identification of transcripts at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. The current study had two aims. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, namely, metaphase II-stage oocytes (MPII), as well as 2-cell, precompact morula (PCM) and in vitro-produced blastocyst (IVTBL) stage embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro- (IVTBL), and nuclear transfer-derived (NTBL) blastocysts. It was hypothesized that the identification of differentially represented transcripts from these embryos would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine expressed sequence tag (EST) library (http://genome.rnet.missouri.edu/bovine/) of female reproductive tissues and embryos were compared using Fisher's Exact Test weighted by number of transcripts per tissue by gene. Of the 3,144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < or = 0.01) in at least one pairwise comparison. Fifteen of these transcripts were selected for further examination using quantitative real-time PCR (qRTPCR) to determine differences in transcript abundance. Twelve of the 15 transcripts were differentially represented (n = 9, P < or = 0.01; n = 3, P < or = 0.05) in at least one pairwise comparison. In summary, identification of differentially represented transcripts in early embryo development, which are modulated by in vitro techniques, should provide markers to ensure the production of embryos closer to those developed in vivo.

Timed artificial insemination of two consecutive services in dairy cows using prostaglandin F2alpha and gonadotropin-releasing hormone.

Timed artificial insemination (TAI) protocols use PGF(2alpha) and GnRH injections to synchronize ovulation. The objective was to evaluate the PGPG protocol (d 0, PGF(2alpha); d 3, GnRH; d 11, PGF(2alpha); d 13, GnRH and TAI) for first TAI and also examine methods for second TAI in nonpregnant cows. A factorial test of the first PGF(2alpha) and first GnRH injections within the PGPG protocol was performed (the last PGF(2alpha) and GnRH injections were deemed essential to the TAI). Lactating dairy cows (n = 804) in a commercial herd were assigned to 1 of 5 first-TAI treatments, which were PGPG, GPG (d 0, no treatment; d 3, GnRH; d 11, PGF(2alpha); d 13, GnRH and TAI), PPG (d 0, PGF(2alpha); d 3, no treatment; d 11, PGF(2alpha); d 13, GnRH and TAI), and PG (d 0, no treatment; d 3, no treatment; d 11, PGF(2alpha); d 13, GnRH and TAI); the Ovsynch protocol (GnRH, 7 d, PGF(2alpha), 2 d, GnRH and TAI) was the positive control. For resynchronization, cows received either GnRH or the control (no injection) on d 22 after TAI. Nonpregnant cows on d 28 were then treated with PGF(2alpha) on d 29, GnRH on d 31, and TAI [i.e., resynchronization treatments of ReGPG (received GnRH on d 22) and RePG (did not receive GnRH on d 22)]. Pregnancy rates for PGPG, GPG, PPG, PG, and Ovsynch were similar at d 28 after first TAI. Analyses of multiple explanatory factors by logistic regression detected an effect of uterine or ovarian abnormality on the d-28 pregnancy rate (normal more likely to be pregnant). Day-42 pregnancy rates were affected by uterine or ovarian abnormality (normal more likely to be pregnant), postpartum disease occurrence (healthy cows more likely to be pregnant), milk production, and days in milk. Treatment was not significant for the d-42 pregnancy rate. Effects of postpartum disease, milk production, and days in milk on the d-42 pregnancy rate were apparently manifested through their effects on embryonic loss between d 28 and 42 of pregnancy. High-producing cows that received TAI early postpartum were most likely to experience embryonic loss. Day-42 pregnancy rates after the resynchronization treatment were affected by an interaction of the first synchronization treatment with the resynchronization treatment. We concluded that although PGPG can be used for TAI, a simpler TAI protocol that includes the last 2 injections (PGF(2alpha), 2 d; GnRH and TAI) would be equally effective.

Parthenogenetic activation of pig eggs by exposure to protein kinase inhibitors.

The objective of the present study was to assess the effect of low concentrations of protein kinase inhibitors on activation. Pig eggs were electrostimulated or cultured with the following: 10 microM 1-[5-isoquinolinylsulfonyl]-2-methylpiperazine, HCl H7 for 24 h; 100 microM H7 for 24 h; 10 nM staurosporine for 24 h; or with 20 microM staurosporine for 20 min followed by Whitten's medium for 24 h. Rates of pronuclear formation in eggs (n = 1240) subjected to these treatments were: untreated, 6.2%; electrostimulated, 77.1%; 10 microM H7, 10.0%; 100 microM H7, 65%; 10 nM staurosporine, 24.2%; and 20 microM staurosporine, 67.3% (significance at P < or = 0.05: 10 microM H7 vs untreated, not significant; 20 microM staurosporine vs 100 microM H7, not significant). Percentages of eggs (n = 125) expressing a 22-kDa band after treatment were: untreated, 37.5%; electrostimulated, 100%; 10 microM H7, 72%; 100 microM H7, 66.7%; 10 nM staurosporine, 40.0%; and 20 microM staurosporine, 77.3% (significance at P < or = 0.10: 100 microM H7, 10 nM staurosporine and 20 microM staurosporine vs 10 microM H7, not significant; 100 microM H7 and 10 nM staurosporine vs untreated, not significant). Transmission electron microscopy of ultrathin sections of treated eggs revealed that cortical granules were present in over half the untreated eggs, as well as over half of the eggs treated with 100 microM H7 or 10 nM staurosporine; in contrast, all cortical granules were absent from electrically-activated eggs. The results indicate that long-term exposure of eggs to low concentrations of broad-spectrum protein kinase inhibitors induces some of the events commonly associated with fertilization.

Genetic and environmental determinants of interferon-tau secretion by in vivo- and in vitro-derived bovine blastocysts.

Several experiments were conducted to assess the effects of genotype and various culture media on interferon-tau secretion by in vitro-derived bovine blastocysts and to compare these values with interferon released by blastocysts flushed from superovulated cows. In experiment 1, oocytes were inseminated with semen from three different bulls. While paternal genotype had no effect on cleavage rate, the size or hatching ability of blastocysts, it was a significant determinant of the embryo's ability to develop to the blastocyst stage and of subsequent interferon-tau secretion. In the second experiment, embryos were cultured in synthetic oviductal fluid containing either polyvinyl alcohol, bovine serum albumin or fetal bovine serum. While there was no effect of supplement on the percentage of embryos developing to the blastocyst stage, blastocysts which formed in medium with polyvinyl alcohol had significantly fewer cells, were older at blastocyst formation and produced significantly more interferon-tau. In the third experiment, embryos were cultured to the blastocyst stage in either TCM199 alone or in co-culture with buffalo rat liver, bovine oviductal or bovine uterine epithelial cells. Culture with oviductal or buffalo rat liver cells increased blastocyst cell number, although secretion of interferon-tau was not affected. In the final experiment, bovine blastocysts were flushed from superovulated cows on Day 7 following insemination. Overall, secretion of interferon-tau by in vivo-produced blastocysts did not differ from that of age-matched blastocysts produced in vitro.

Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor.

This study examined the ability of epidermal growth factor (EGF) to improve the developmental competence of pig oocytes matured in a protein-free (PF) in vitro maturation (IVM) system. Oocyte maturation was done in one of three media: 1. PF-TCM: tissue culture medium (TCM) 199 + 0.1% polyvinylalcohol (PVA); 2. PF-TCM+EGF: PF-TCM + 10 ng/ml EGF; and 3. +ve CONT: North Carolina State University (NCSU) 23 medium + 10% porcine follicular fluid. All media contained 0.57 mM cysteine. Hormonal supplements, 0.5 microg/mL LH and 0.5 microg/mL FSH, were present only for the first half (20 to 22 h) of the culture period. After maturation, oocytes were co-incubated with frozen-thawed spermatozoa for 5 to 6 h and transferred to embryo culture medium, NCSU 23 containing 0.4% BSA, for 144 h. In Experiment 1, differences in cumulus expansion were observed for oocytes matured in +ve CONT (Category 4), PF-TCM (Category 2) and PF-TCM+EGF (Category 3). However, no significant differences in nuclear maturation to metaphase II stage were observed. In Experiment 2, no differences in fertilization parameters were observed. Significant (P < 0.01) differences in cleavage rates were observed among the three media for a proportion of the oocytes matured (52, 60 and 69% in PF-TCM, PF-TCM+EGF, and +ve CONT, respectively). Oocytes matured in PF-TCM showed the lowest (P < 0.01) blastocyst development (22%). However, the same rate of blastocyst development was obtained for +ve CONT (37%) and PF-TCM+EGF (37%). Blastocyst cell numbers were significantly higher when oocytes were matured in the presence of EGF (26 vs. 37 to 41). In Experiment 3, oocytes matured in PF-TCM+EGF had a significantly (P < 0.05) higher intracellular glutathione (GSH) concentration (5.9 vs. 11.4 pmol/oocyte) compared with PF-TCM. Twenty-two of 25 embryo transfer recipients became pregnant (Experiment 4). Four animals returned to estrus in within 60 days. Six pregnant animals slaughtered at 26 to 45 days had 43 fetuses (range: 4 to 12) and the remaining 12 animals farrowed 82 piglets (range: 3 to 12). These results indicate that EGF enhances the developmental competence of pig oocytes matured in a protein-free culture medium which is correlated with higher GSH level in oocytes. Birth of piglets indicate that embryos derived from oocytes matured in the presence of EGF are viable.

Plasma GH, IGF-I, and conception rate in cattle treated with low doses of recombinant bovine GH.

Blood and uterine concentrations of GH and insulin-like growth factor (IGF)-I are correlated with improved fertility in cattle. We tested incremental doses of a 14-d sustained release recombinant bovine GH (rbGH) to increase blood GH and IGF-I (Experiments 1 and 2). Conception rate after administration of an optimized rbGH dose was also tested (Experiment 3). In Experiment 1, lactating Holstein cows (n = 18) were randomly assigned to receive 0 (n = 5), 100 (n = 5), 200 (n = 5), or 500 (n = 3) mg sc rbGH. Increasing the doses of rbGH was associated with increased serum concentrations of GH and IGF-I. The 100- and 200-mg doses caused an IGF-I release that was below and above, respectively, the perceived optimum response. Therefore, Experiment 2 was designed to test a rbGH dose (167 mg), which was intermediate to the doses tested in Experiment 1. Lactating and nonlactating postpartum beef cows were treated with 0 (n = 9) or 167 (n = 9) mg rbGH at insemination. Plasma concentrations of GH and IGF-I were greater in rbGH-treated cows than in controls. Lactating cows had initial IGF-I concentrations that were lower than nonlactating cows. The 167-mg dose of rbGH increased plasma IGF-I concentrations in lactating cows to the levels of those of nonlactating cows. In Experiment 3, cows and heifers were administered either 0 or 167 mg rbGH at insemination. The conception rate for rbGH-treated and control cows was 54.4 and 49.5% (n = 617), and 46.0 and 46.3% for heifers (n = 1123), respectively. Herd (P<0.01) and parity (P<0.01) affected conception rate, but conception rates for rbGH and control cattle were similar. In summary, low doses of rbGH increased blood GH and restored blood IGF-I concentrations in lactating cows to those of nonlactating cows, but the conception rate in cows and heifers was not affected by administration of 14-d sustained-release rbGH at insemination.

Heat tolerance in two tropically adapted Bos taurus breeds, Senepol and Romosinuano, compared with Brahman, Angus, and Hereford cattle in Florida.

Two trials were conducted with heifers to determine heat tolerance among temperate Bos taurus (Angus, Hereford), Bos indicus (Brahman), tropical Bos taurus (Senepol, Romosinuano), and the reciprocal crosses of Hereford and Senepol. Differences among breeds in temperament score, circulating concentrations of cortisol, and blood packed cell volume were also investigated. Trial 1 used 43 Angus, 28 Brahman, 12 Hereford, 23 Romosinuano, 16 Senepol, 5 Hereford x Senepol (H x S), and 5 Senepol x Hereford (S x H) heifers. Trial 2 used 36 Angus, 31 Brahman, 9 Hereford, 14 Senepol, 19 H x S, and 10 S x H heifers. On the hottest summer date in Trial 1, rectal temperature of Angus was greater (P < .001) than that of Brahman, Senepol, or Romosinuano. Rectal temperature and plasma cortisol were significantly less in Senepol than in Brahman, suggesting that the differences in rectal temperature between these breeds may be due to differences in stress response possibly related to differences in temperament. Reciprocal crosses of Hereford and Senepol had rectal temperatures nearly as low as that of Senepol and displayed substantial heterosis (-9.4%, P < .05) in log10 rectal temperature on the hottest summer date. On both the hottest and coolest dates in Trial 1, Angus heifers had significantly faster respiration rates than Brahman, Romosinuano, or Senepol heifers, and Brahman had significantly slower respiration rates than Romosinuano or Senepol. On the hottest summer date in Trial 2, rectal temperature in Angus heifers was greater (P < .001) than in Brahman or Senepol had rectal temperatures similar to that of Senepol, or heterosis for log10 rectal temperature was similar to that in Trial 1 (-9.8%, P < .05). Considering rank order among breeds, Brahman always had the slowest respiration rate and greatest packed cell volume. Brahman had significantly greater temperament scores and plasma cortisol concentrations than Angus or Senepol, except that plasma cortisol was not different between Brahman and Senepol on the hottest summer date. On this date, rectal temperature did not differ between Brahman and Senepol, which supports the hypothesis that there is a relationship between response to stress and rectal temperature that helps explain differences in rectal temperature between Brahman and Senepol. The results of these trials demonstrate heat tolerance of the Senepol and Romosinuano, two Bos taurus breeds. Furthermore, the results suggest a substantial level of dominance of the Senepol's ability to maintain constant body temperature in a hot environment as measured by rectal temperature in crosses with a non-adapted breed.

Field experience trials comparing narasin and monensin.

Narasin is effective against all species of chicken coccidia when tested in battery cage and floor pen studies. To confirm the efficacy of narasin under practical broiler production conditions, the drug was fed at concentrations of 60 ppm or 80 ppm to broiler chickens being raised by six different commercial broiler producers in five different geographic areas. Monensin was fed in each trial at a concentration of 100 ppm or 121 ppm as a reference control. The usual management practices of each of the integrated broiler companies were followed throughout the respective trials. Nine trials were conducted and approximately 100,000 broilers were tested for each treatment. No adverse reactions attributable to treatments were observed in any of the trials, and performance results obtained with narasin-medicated birds were generally comparable with those obtained with monensin-medicated birds in the same trial. These findings support the conclusion that narasin at final feed concentrations in the range of 60 to 80 ppm can be safely and effectively used as an anticoccidial agent in commercial broiler production facilities.

Birth of piglets from in vitro-produced, zona-intact porcine embryos vitrified in a closed system.

As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN(2)). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a "closed" system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease.

Porcine oocytes denuded before maturation can develop to the blastocyst stage if provided a cumulous cell-derived coculture system.

The physiological role of cumulus cells (CC) surrounding oocytes is particularly important for normal cytoplasmic maturation of oocytes. However, removal of CC from oocytes is inevitable for some embryo manipulation techniques, such as germinal vesicle (GV) transfer, somatic cell haploidization, and oocyte cryopreservation. The present study was designed to determine an optimal method to culture porcine denuded oocytes (DO). The results indicated CC from cumulus-oocyte complexes at the GV stage (GVCC) or at the metaphase II stage, and mural granulosa cells could not improve the maturation of DO. However, GVCC could enhance the development of matured porcine DO after fertilization; the percentage of blastocysts was increased from 1.1 to 17.2% (P < 0.05), and the relative value of the x-axis and y-axis of spindles was also increased (P < 0.05). Coculture with GVCC had no effect on the distribution of mitochondria and cortical granules. The results contribute to our understanding of the mechanisms by which CC promote oocyte maturation and contribute to optimization of protocols for in vitro maturation of DO.

Production of endothelial nitric oxide synthase (eNOS) over-expressing piglets.

Vascular function, vascular structure, and homeostasis are thought to be regulated in part by nitric oxide (NO) released by endothelial cell nitric oxide synthase (eNOS), and NO released by eNOS plays an important role in modulating metabolism of skeletal and cardiac muscle in health and disease. The pig is an optimal model for human diseases because of the large number of important similarities between the genomic, metabolic and cardiovascular systems of pigs and humans. To gain a better understanding of cardiovascular regulation by eNOS we produced pigs carrying an endogenous eNOS gene driven by a Tie-2 promoter and tagged with a V5 His tag. Nuclear transfer was conducted to create these animals and the effects of two different oocyte activation treatments and two different culture systems were examined. Donor cells were electrically fused to the recipient oocytes. Electrical fusion/activation (1 mM calcium in mannitol: Treatment 1) and electrical fusion (0.1 mM calcium in mannitol)/chemical activation (200 microM Thimerosal for 10 min followed by 8 mM DTT for 30 min: Treatment 2) were used. Embryos were surgically transferred to the oviducts of gilts that exhibited estrus on the day of fusion or the day of transfer. Two cloned transgenic piglets were born from Treatment 1 and low oxygen, and another two from Treatment 2 and normal oxygen. PCR, RT-PCR, Western blotting and immunohistochemistry confirmed that the pigs were transgenic, made message, made the fusion protein and that the fusion protein localized to the endothelial cells of placental vasculature from the conceptuses as did the endogenous eNOS. Thus both activation conditions and culture systems are compatible with development to term. These pigs will serve as the founders for a colony of miniature pigs that will help to elucidate the function of eNOS in regulating muscle metabolism and the cardiorespiratory system.

Immunocytochemical analysis of the association of bovine oviduct-specific glycoproteins with early embryos.

The bovine oviductal epithelium synthesizes and secretes a class of oviduct-specific glycoproteins that is present in the luminal fluid when fertilization and early embryonic development occur. The objective of this study was to determine if these characterized glycoproteins become associated with oviductal embryos. Ovarian ova and oviductal embryos were recovered from super-ovulated cows at 72 h after onset of estrus. Eggs were fixed in 3% paraformaldehyde-1% glutaraldehyde and subsequently embedded in Lowicryl K4M. Sections (1 micron) were processed for peroxidase-antiperoxidase immunocytochemistry. Immunolabeling was not detected in any region of ovarian ova. Oviductal embryos, regardless of cleavage stage, exhibited immunoperoxidase staining localized within their zona pellucidae. Sections (100 nm) obtained from a 4- and an 8-cell embryo were also subjected to colloidal gold immunoelectron microscopy to determine conclusively the subcellular distribution of the oviduct-specific glycoproteins. Gold particles were distributed uniformly throughout the width of the zona pellucida. Also, immunoreactivity was observed associated with flocculent material in the perivitelline space and with the vitelline membrane. These results indicate that the bovine oviduct-specific secretory glycoproteins become associated with oviductal embryos. This association may be biologically important to the developing embryo.

Induction of trophoblastic interferon expression in ovine blastocysts after treatment with double-stranded RNA.

Ovine trophoblast protein-1 (oTP-1) is an interferon (IFN) related to the IFN-omega. The objectives of this research were: (i) to attempt to induce oTP-1 mRNA in day-11 ovine conceptuses with polyinosinic-polycytidylic acid (poly(I).poly(C], and (ii) to determine if IFN-omega mRNA is also produced on day 11 of gestation. In experiment I, conceptuses were cultured in presence of 100 micrograms/ml poly(I).poly(C) (n = 5) or medium alone (control, n = 3) for up to 8 h. In situ hybridization was used to assess effects of treatment on mRNA concentrations for oTP-1 and actin (positive hybridization control). Poly(I).poly(C) increased oTP-1 mRNA concentrations approximately 2.5-fold (p less than 0.01), but had no effect on actin mRNA. In experiment II, the presence of mRNA for oTP-1 and ovine IFN-omega was determined by using reverse transcription-polymerase chain reaction (RT-PCR) analysis of conceptus total RNA coupled with Southern blot hybridization of the PCR reaction products with specific cDNA probes. oTP-1 mRNA was detectable in all poly(I).poly(C)-treated (n = 7) and control (n = 6) conceptuses, whereas IFN-omega mRNA was detected in only three of seven poly(I).poly(C)-treated conceptuses and not in any controls. Together these results demonstrate that expression of oTP-1 mRNA can be enhanced by treatment with poly(I).poly(C) and that oTP-1 is the primary but not the only type I-IFN inducible in conceptuses on day 11 of gestation.

Expression of bovine trophoblast interferon in conceptuses derived by in vitro techniques.

Expression of the trophoblast interferon, bovine trophoblast protein-1 (bTP-1), has been studied in embryos produced by in vitro maturation-in vitro fertilization (IVM-IVF). No bTP-1 production was noted until after embryos had reached the expanded blastocyst stage and had begun to hatch (Days 8-9 post-fertilization). Single blastocysts comprising 115 +/- 22 cells released 1.0 +/- 0.1 units of interferon activity/24 h. Amplification of conceptus mRNA by reverse transcription-polymerase chain reaction procedure with bTP-1-specific oligonucleotides confirmed that bTP-1 transcripts were present in blastocysts but were not detectable at earlier stages. Although cultured blastocysts produced by IVM-IVF procedures continued to secrete bTP-1 for a few days, they failed to attach to the substratum and form outgrowths, and soon lost structural integrity. However, when Day 8 blastocysts/morulae were transferred to the uteri of synchronized cows, recovered 4 days later, and placed into individual cultures, they attached and formed outgrowths that produced large amounts of bTP-1 (greater than 2000 units/culture/24 h after 14 days). Embryos thus first expressed bTP-1 when a functional trophectoderm was first formed, and induction did not require a period of in vivo development. However, continued viability of the blastocyst and bTP-1 production were not sustained in vitro and may require some exposure to the uterine environment.

Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes.

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8+/-17.3% vs 28.5+/-3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0+/-7.0% vs 63.3+/-12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0+/-11.6% vs 4.6+/-4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.