The healthy equine uterus harbors a distinct core microbiome plus a rich and diverse microbiome that varies with geographical location.

The goal of this study was to understand the composition and existence of the resident uterine microbiome in healthy mares and to establish the presence of a core microbiome for the healthy equine uterus. We analyzed the microbiomes of 35 healthy mares that are long-time residents of three farms in Oklahoma, Louisiana, and Australia as well as that of 19 mares purchased from scattered owners in the Southern Mid-Western states of the United States. Over 6 million paired-end reads of the V4 region of the 16S rRNA gene were obtained resulting in 19,542 unique Amplicon Sequence Variants (ASVs). ASVs were assigned to 17 known phyla and 213 known genera. Most abundant genera across all animals were Pseudomonas (27%) followed by Lonsdalea (8%), Lactobacillus (7.5%), Escherichia/Shigella (4.5%), and Prevotella (3%). Oklahoma and Louisiana samples were dominated by Pseudomonas (75%). Lonsdalea (28%) was the most abundant genus in the Australian samples but was not found in any other region. Microbial diversity, richness, and evenness of the equine uterine microbiome is largely dependent on the geographical location of the animal. However, we observed a core uterine microbiome consisting of Lactobacillus, Escherichia/Shigella, Streptococcus, Blautia, Staphylococcus, Klebsiella, Acinetobacter, and Peptoanaerobacter.

Canine endometrial and vaginal microbiomes reveal distinct and complex ecosystems.

The objective of this study was to characterize the normal microbiome of healthy canine vagina and endometrium and to determine the effect of the stage of estrous, on the resident microbiome. Cranial vaginal swabs and uterine biopsy samples were collected from twenty-five bitches in five different stages of estrous at elective ovariohysterectomy (OVH). Over 4 million reads of the V4 region of 16S rDNA gene were obtained and used for further analyses. A total of 317 genera belonging to 24 known phyla were identified. The endometrium was higher in bacterial diversity while the vagina was higher in richness. Proteobacteria, Bacteroidetes and Firmicutes were the most abundant phyla observed across all samples. Hydrotalea, Ralstonia, and Fusobacterium accounted for nearly 60% of the OTUs identified in the vagina while organisms identified in the endometrium were more evenly distributed. Pseudomonas, Staphylococcus, and Corynebacterium were the prominent genera in the endometrium. The microbiome of the endometrium was distinctly different from that of the vagina. There was large animal-to-animal variation. Other than the vaginal microbiome of bitches in estrus (i.e. in heat), there were no distinct clustering of the organisms based on the stage of estrous. These findings establish the presence of a resident microbiome of the endometrium throughout all stages of estrous cycle.

Integrative veterinary medical education and consensus guidelines for an integrative veterinary medicine curriculum within veterinary colleges.

Integrative veterinary medicine (IVM) describes the combination of complementary and alternative therapies with conventional care and is guided by the best available evidence. Veterinarians frequently encounter questions about complementary and alternative veterinary medicine (CAVM) in practice, and the general public has demonstrated increased interest in these areas for both human and animal health. Consequently, veterinary students should receive adequate exposure to the principles, theories, and current knowledge supporting or refuting such techniques. A proposed curriculum guideline would broadly introduce students to the objective evaluation of new veterinary treatments while increasing their preparation for responding to questions about IVM in clinical practice. Such a course should be evidence-based, unbiased, and unaffiliated with any particular CAVM advocacy or training group. All IVM courses require routine updating as new information becomes available. Controversies regarding IVM and CAVM must be addressed within the course and throughout the entire curriculum. Instructional honesty regarding the uncertainties in this emerging field is critical. Increased training of future veterinary professionals in IVM may produce an openness to new ideas that characterizes the scientific method and a willingness to pursue and incorporate evidence-based medicine in clinical practice with all therapies, including those presently regarded as integrative, complementary, or alternative.

Transrectal ultrasound-integrated spectral optical tomography of hypoxic progression of a regressing tumor in a canine prostate.

The objective of this study was to evaluate if transrectal optical tomography implemented at three wavelength bands for spectral detection could monitor changes of the hemoglobin oxygen saturation (StO2) in addition to those of the total hemoglobin concentration ([HbT]) in lesions of a canine prostate, including an induced tumor modeling canine prostate cancer. Near-infrared (NIR) optical tomography was integrated with ultrasound (US) for transrectal imaging. Multi-spectral detection at 705_nm, 785_nm and 808_nm rendered measurements of [HbT] and StO2. Canine transmissible venereal tumor (TVT) cells were injected into the right lobe of a dog's prostate gland, which had a pre-existing cyst in the left lobe. Longitudinal assessments of the prostate were performed weekly over a 63-day duration by NIR imaging concurrent with grey-scale and Doppler US. Ultrasonography revealed a bi-lobular tumor-mass regressing from day-49 to day-63. At day-49 this tumor-mass developed a hypoxic core that became larger and more intense by day-56 and expanded further by day-63. The tumor-mass presented a strong hyper-[HbT] feature on day-56 that was inconsistent with US-visualized blood flow. Histology confirmed two necrotic TVT foci within this tumor-mass. The cyst appeared to have a large anoxic-like interior that was greater in size than its ultrasonographically delineated lesion, and a weak lesional elevation of [HbT]. On day-56, the cyst presented a strong hyper-[HbT] feature consistent with US-resolved blood flow. Histology revealed acute and chronic hemorrhage in the periphery of the cyst. The NIR imaging features of two other TVT nodules and a metastatic lymph node were evaluated retrospectively. Transrectal US-integrated spectral optical tomography seems to enable longitudinal monitoring of intra-lesional oxygenation dynamics in addition to the hemoglobin content of lesions in the canine prostate.

Equine endometrial biopsy reviewed: observation, interpretation, and application of histopathologic data.

The equine endometrial biopsy, an important tool in equine reproduction science, has experienced a rich period of increasing knowledge, development, and application over the past 40 y. Much of the foundational work in this field was conducted by Dr. Robert M. Kenney. In view of his recent passing, this review is dedicated to our alumnus, Dr. Robert M. Kenney (OSU, 1954). In this manuscript, we pay tribute to Kenney-eponymous for the equine endometrial biopsy grading system-by reviewing the procedure. We present this review in three parts: 1) how observational data are acquired; 2) how these data are interpreted; and 3) how these data are applied in equine reproduction science and medicine.

Infection of embryos following insemination of donor mares with equine arteritis virus infective semen.

The objective was to evaluate the potential risks associated with embryo transfer from mares bred with equine arteritis virus (EAV) infective semen. Twenty-six mares were embryo donors, whereas 18 unvaccinated and EAV antibody seronegative mares were embryo recipients. Of the 26 donor mares, 15 were unvaccinated and seronegative for antibodies to EAV and 11 were vaccinated for the first time with a commercially available modified live virus vaccine against EVA before breeding and subsequent embryo transfer. All donor mares were bred with EAV-infective semen from a stallion persistently infected with the virus. Twenty-four embryos were recovered 7 d post-ovulation; all were subjected in sequential order to five washes in embryo flush medium, two trypsin treatments, and five additional washes in embryo flush medium (prior to transfer). Twelve and seven embryos (Grades 1 or 2) were transferred from the non-vaccinated and vaccinated donors, respectively, and pregnancy was established in 3 of 12 and 2 of 7. Perhaps trypsin reduced embryo viability and pregnancy rate. The uterine flush fluid of 11 mares (9 of 15 and 2 of 11 from non-vaccinated and vaccinated donor groups, respectively) was positive for EAV by VI (confirmed by real-time RT-PCR); the wash fluid from the embryos of nine of these mares was negative following 10 washes and two trypsin treatments. However, the embryo wash fluid from two mares was still positive for EAV after all 10 washes and the two trypsin treatments, and one embryo was positive for EAV. Two of 18 recipient mares had seroconverted to EAV 28 d after embryo transfer. Virus was not detected in any fetal tissues or fluids harvested after pregnancies were terminated (60 d). In conclusion, we inferred that the washing protocol of 10 washes and two trypsin treatments did not eliminate EAV from all embryos; due to limitations in experimental design, this requires confirmation. Furthermore, there may be a risk of EAV transmission associated with in vivo embryo transfer from a donor mare inseminated with EAV infective semen.

Trans-rectal ultrasound-coupled spectral optical tomography of total hemoglobin concentration enhances assessment of the laterality and progression of a transmissible venereal tumor in canine prostate.

OBJECTIVES: To evaluate whether trans-rectal spectral optical tomography of total hemoglobin concentration (HbT) can image longitudinal and lateral developments of a canine transmissible venereal tumor (TVT) in a canine prostate. METHODS: A near-infrared (NIR) applicator was integrated with a trans-rectal ultrasound (TRUS) transducer to perform ultrasound (US)-coupled optical tomography of the canine prostate. Spectral detection at 785 and 830 nm enabled quantitation of HbT. Canine TVT cells were injected into the right lobe of a dog's prostate gland. Longitudinal imaging assessment of the post-injection prostate was performed by coupled US/NIR imaging over a 45-day duration. RESULTS: By day 7, NIR indicated TVT infiltration in the noninjected left prostatic lobe with the gray-scale US indistinct. By day 31, both NIR and gray-scale US revealed more widespread TVT involvement in the left than in the right lobe, as well as an extensive TVT mass in the caudal aspect of the gland, of which the peak HbT increased 3-fold and the mass volume grew exponentially over the 45-day duration. Increased blood supply to the mass was also observed on Doppler US. CONCLUSIONS: TRUS-coupled spectral optical tomography enhances assessment of the laterality and progression of prostate tumor compared with using gray-scale and Doppler TRUS.

Bovine viral diarrhea virus abortion in goats housed with persistently infected cattle.

Twenty-four border disease virus-seronegative, pregnant, mixed breed goats were experimentally comingled with 3 heifers persistently infected with bovine viral diarrhea virus type 2a (BVDV-2a). Twelve of the 24 exposed does aborted. Twenty-nine fetuses and 16 placentas from affected does were submitted to the Oklahoma Animal Disease Diagnostic Laboratory for a necropsy examination. Infection with BVDV was confirmed with a combination of immunohistochemistry, BVDV-2 polymerase chain reaction, and virus isolation in 19 of the 29 fetuses. On gross examination of the 19 fetuses and placentas in which BVDV-2a infection was confirmed, a mild placentitis (3/19), fetal mummification (1/19), and facial deformities (4/19) were noted. Histologically, placentitis (2/19), myocarditis (4/19), thymic depletion (5/19), choroid plexitis (3/19), encephalitis (2/19), and cerebral gliosis (1/19) were noted. Other causes of abortion in goats, including common bacterial and viral infections, were ruled out with histology, virus isolation, polymerase chain reaction, and aerobic bacteriologic cultures. As supported by the findings in this case, BVDV-2a should be included as a differential for abortion in goats. This is the first report of abortion in goats after exposure to persistently infected cattle.

Distribution of bovine viral diarrhea virus antigen in aborted fetal and neonatal goats by immunohistochemistry.

Bovine viral diarrhea virus (BVDV) infection in goats can result in severe reproductive losses, with abortion rates reaching 80%. Infection with BVDV in aborted goat fetuses and stillborn kids can result in placentitis, encephalitis, myocarditis, and thymic depletion. This study investigates the distribution of viral antigen within the organ systems of aborted goat fetuses, stillborn kids, and nonviable kids infected with BVDV at various stages of gestation using immunohistochemistry (IHC). Virus antigen was detected within the placenta (8/13), thymus (4/9), heart (4/11), and brain (4/15) of affected goats. Uncommonly, BVDV antigen was detected within the skin (1/14), liver (1/13), kidney (1/12), lung (1/11), and trachea (1/3). BVDV antigen was not detected within the spleen (0/9), nasal turbinate (0/2), or thyroid (0/3). The results of this study indicate that placenta, heart, thymus, and brain are the most reliable tissues for BVDV antigen detection using IHC in aborted goat fetuses.

Risks of transmitting ruminant spongiform encephalopathies (prion diseases) by semen and embryo transfer techniques.

Early experiments suggested that scrapie transmission via sheep embryos was a possibility, and gave rise to much controversy. However, when account is taken of the complex genetic effects on ovine susceptibility to scrapie, and of the several different scrapie strains with different clinical and pathological effects, the overall conclusion now is that transmission of classical scrapie by embryo transfer is very unlikely if appropriate precautions are taken. Recent embryo transfer studies have confirmed this. Other studies in sheep have shown that from about the middle of pregnancy the placental trophoblast is liable to scrapie infection in genetically susceptible ewes if the fetus is also susceptible. Since the contrary is also true, use of resistant ewes as embryo recipients could add to the safety of the embryo transfer, at least for classical scrapie. There has been little recent research on scrapie transmission via semen in sheep, and, with hindsight, the early studies, though negative, were inadequate. There is scant information on scrapie transfer via goat semen or embryos, although one study did find that bovine spongiform encephalopathy (BSE) was not transmitted via goat embryos. In cattle it has been shown that, if appropriate precautions are taken, the risks of transmitting BSE via semen and in vivo-derived embryos are negligible, and this conclusion has gained worldwide acceptance. Research on TSE transmission via reproductive technologies in deer has not yet been done, but information on the pathogenesis and epidemiology of chronic wasting disease (CWD) of deer, and on transmission risks in other species, provides optimism that transmission of CWD via semen and embryos of deer is unlikely. The presence of TSE infectivity in blood and various other tissues of infected animals, particularly sheep, gives rise to concerns that certain biological products currently used in reproductive technologies, e.g. pituitary gonadotrophins for superovulation, and certain tissue and blood products used in semen and embryo transfer media, could carry TSE infectivity. Instruments such as laparoscopes used for insemination, and for collection and transfer of embryos, especially in small ruminants, are also a concern because effective decontamination can be very difficult.

Equine viral arteritis: current status and prevention.

Recently, there has been increased interest in equine viral arteritis (EVA) among veterinarians and horse owners. Outbreaks of the disease were identified initially in New Mexico, USA in 2006, and in the Normandy region of France in the summer of 2007. Both occurrences were associated with AI of cool-shipped semen. Each was linked to respiratory illness, neonatal death, abortion, development of carrier stallions, and cancellation of equestrian events. In light of the increased interest, this paper will present a brief case history, followed by a review addressing common concerns regarding EVA, current status, and control and prevention strategies, including vaccination, and recommended bio-security measures.

Serum antibodies in mares and foals to Actinobacillus equuli whole cells, outer membrane proteins, and Aqx toxin.

Actinobacillus equuli is carried in the alimentary tract of mares and can cause severe septicemia of neonatal foals. A hemolytic subspecies, A. equuli subsp. haemolyticus, and a non-hemolytic subspecies, A. equuli subsp. equuli, have been identified. Hemolytic strains produce the RTX toxin Aqx. The purpose of this study was to demonstrate sequentially in two sets of mare-foal pairs antibodies to A. equuli whole bacterial cells, outer membrane proteins, and recombinant Aqx and to compare the transfer of antibodies to these antigens between mares and their foals. Two mare/foal sets of sera were evaluated. Cohort A consisted of 18 mare-foal pairs obtained in the spring of 2005. Cohort B consisted of 10 mare-foal pairs obtained in the spring of 2006. For both sets, mare and foal sera were obtained immediately after foaling and prior to nursing (time 0) as well as at 12 and 24h and daily thereafter for 7 days. For Cohort B, sera were also obtained 30 days after birth. At parturition all mares had detectable antibodies to A. equuli whole cells and outer membranes; however, of those mares, two in Cohort A had undetectable antibodies to Aqx and their foals likewise had undetectable anti-Aqx antibodies. Antibodies against whole cells, outer membrane proteins, and Aqx were readily transferred from mares to foals. In most cases, there were significant correlations (p<0.05) between antibodies against whole cells, outer membrane proteins, and Aqx in mares' sera at the time of parturition and foal sera 24 after birth. Antibodies against the three antigen preparations had declined insignificantly (p>0.05) by day 30.

Association between blood plasma urea nitrogen levels and reproductive fluid urea nitrogen and ammonia concentrations in early lactation dairy cows.

Two experiments were conducted to study the relationship of blood plasma urea nitrogen (PUN) concentrations with NH3, urea nitrogen, K, Mg, P, Ca, and Na concentrations in fluid of preovulatory follicles (experiment 1) and the relationships of PUN concentration and stage of estrus cycle with ammonia and urea nitrogen concentrations in uterine fluids (experiment 2) in early lactation dairy cows. Mean PUN levels were used to distribute cows into two groups: cows with PUN>or=20 mg/dl (HPUN), and cows with PUN<20 mg/dl (LPUN). In experiment 1, blood and follicular fluids from preovulatory follicles of 38 early lactation dairy cows were collected on the day of estrus (day 0) 4h after feed was offered. Follicular fluid NH3 was higher (P<0.01) in HPUN cows (339.0 micromol/L+/-72.2) compared to LPUN cows (93.9 micromol/L+/-13.1). Follicular fluid urea N was higher (P<0.001) in HPUN cows (22.4 mg/dl+/-0.4) compared to LPUN cows (17.0 mg/dl+/-0.3). PUN and follicular fluid urea N were correlated (r2=0.86) within cows. In experiment 2, blood and uterine fluids were collected from 30 cows on day 0 and on day 7. Uterine fluid NH3 was higher (P=0.05) in HPUN cows (1562 micromol/L+/-202) than in LPUN cows (1082 micromol/L+/-202) on day 7, but not on day 0. Uterine fluid urea N was higher (P<0.001) in HPUN cows than in LPUN cows on day 0 (26.9 mg/dl+/-1.3 and 20.4 mg/dl+/-0.7) and day 7 (26.5 mg/dl+/-1.1 and 21.4 mg/dl+/-1.1). There was a correlation (r2=0.17) between PUN and uterine fluid urea N within cows. The results of this study indicate that high PUN concentrations were associated with elevated NH3 and urea N concentrations in the preovulatory follicular fluids on the day of estrus and in the uterine fluid during the luteal phase of the estrous cycle in early lactation dairy cows. Elevated NH3 or urea N concentrations in the reproductive fluids may contribute to reproductive inefficiency in dairy cows with elevated plasma urea nitrogen due to embryo toxicity.

Preventing experimental vertical transmission of scrapie by embryo transfer.

This study investigated whether the transmission of naturally occurring scrapie in sheep can be prevented using embryo transfer. Embryos were collected from 38 donor ewes in a Suffolk sheep flock with a high incidence of naturally occurring scrapie, treated with a sanitary procedure (embryo washing) recommended by the International Embryo Transfer Society and then transferred to 58 scrapie-free recipient ewes. Ninety-four offspring were produced. None of the offspring or the recipient ewes developed scrapie. Furthermore, offspring derived from embryos collected from donor ewes bred to the immunohistochemically positive ram did not develop scrapie. We conclude that scrapie was not transmitted to offspring via the embryo nor was the infective agent transmitted to recipient ewes during embryo transfer procedures.

The influence of the phase of the estrous cycle on sheep endometrial tissue response to lipopolysaccharide.

The effect of lipopolysaccharide on ovine endometrial tissue was examined at estrus (follicular phase) and during the luteal phase. Endometrial tissues were cultured with 0, 1, or 10 microg/mL lipopolysaccharide. After 24 h, culture supernates were harvested and analyzed for PGF2alpha, PGE2, 6-keto-PGF1alpha, thromboxane B2 (TXB2), and cysteinyl-leukotrienes (leukotrienes) using EIA. Homogenates of endometrial tissue were analyzed for prostaglandin endoperoxidase-1 (PTGS-1), and -2 (PTGS-2) as well as Type-I, -II, and -III nitric oxide synthase (NOS) by Western analysis. Follicular phase tissue produced more PGF2alpha (P < 0.001), TXB2 (P < 0.001), and leukotrienes (P < 0.02) than luteal tissue. Lipopolysaccharide increased PGE2 (P < 0.001) and TXB2 (P < 0.02) production by endometrial tissue. Elevations in these eicosanoids were likely due to the measured increases in PTGS-2 (P = 0.002) because no changes in PTGS-1 (P = 0.54) or Type-I, -II, or -III NOS (P > or = 0.20) occurred in endometrial tissue following lipopolysaccharide exposure. These data suggest that the phase of the estrous cycle regulates prostaglandin production by immune-challenged endometrial tissue.

Effects of ammonia during different stages of culture on development of in vitro produced bovine embryos.

The effects of various concentrations of ammonia in the media during in vitro fertilization (IVF), culture (IVC), and throughout maturation (IVM), IVF, and IVC were evaluated using a randomized complete block design. Ammonia was added to the media at various concentrations during IVF (experiment 1), during IVC (experiment 2), and throughout IVM, IVF, and IVC (experiment 3). In the first experiment, there was a significant (P<0.05) increase in embryos developed to blastocyst, and to expanding and hatching blastocyst, in IVF media containing moderate concentrations of ammonia compared with that in the IVF control media. In the second experiment, ammonia in the IVC media increased (P<0.05) the proportion of degenerate ova and decreased (P<0.05) the proportion of ova that developed to blastocysts. In experiment 3, cleavage rates tended (P=0.06) to be greater for control groups than for treatment groups. The proportion of ova developing to morula was greater (P<0.05) in media containing moderate concentrations of ammonia than that in the control groups. These results indicate that the effect of ammonia on development of preimplantation bovine embryos depends on the concentration of ammonia and the stage of development when exposure to ammonia occurs.

Ammonia concentration in bovine follicular fluid and its effect during in vitro maturation on subsequent embryo development.

10 mm) to determine ammonia concentration. Ammonia concentration was greater (P<0.001) in the bFF from smaller follicles compared with bFF from larger follicles, and ammonia concentration decreased (P10 mm follicles, respectively. In Experiment 2, abattoir oocytes (n=2885) were matured in vitro in media containing ammonia at the concentrations of 0 microM (Control), 29 microM (A1), 88 microM (A2), 132 microM (A3), 176 microM (A4) and 356 microM (A5), followed by in vitro fertilization and culture. There was no difference (P0.05) in cleavage rates and morula or blastocyst development between treatments, indicating that in vitro exposure of bovine oocytes to ammonia does not adversely influence subsequent embryonic development.

The influence of reproductive phase on lipopolysaccharide stimulation of prostacyclin production and cyclooxygenase-2 protein concentrations in reproductive and systemic arteries of the sheep.

Cyclooxygenase, the enzyme that converts arachidonate to prostaglandins, plays a regulatory role in vasodilation under normal and pathological conditions. Studies were conducted to determine the effects of reproductive phase and lipopolysaccharide (LPS) on production of PGI2 and amounts of cyclooxygenase protein in uterine, mammary, mesenteric, and renal arteries. Arteries were collected from ewes during the follicular (Day 0 = estrus) or luteal (Day 10) phase of the estrous cycle and were cultured in the presence of LPS. After 24 h, media were collected and analyzed for 6-keto-PGF1alpha, the stable metabolite of PGI2. In addition, arteries were collected and homogenized and the relative concentration of cyclooxygenase was determined via Western analysis. Lipopolysaccharide stimulated PGI2 production in all four-artery types from both follicular and luteal phase ewes (p < 0.001). Upon LPS stimulation, uterine and mammary arteries produced more PGI2 compared to mesenteric and renal arteries (p = 0.04). The phase of estrous cycle did not affect PGI2 production by any of the artery populations exposed to LPS (p = 0.35). There was no cyclooxygenase-2 in untreated uterine and mammary arteries and no cyclooxygenase-2 was detected in untreated or LPS-treated mesenteric and renal arteries. In contrast, LPS-treated uterine and mammary arteries from luteal phase ewes had higher (p = 0.064) cyclooxygenase-2 concentrations than those from follicular phase ewes. These results suggest that the hormone conditions of the follicular (high estrogen) and luteal (high progesterone) phases of the ovarian cycle play a role in regulating uterine and mammary artery but not mesenteric and renal artery response to LPS.

Cellular source in ewes of prostaglandin-endoperoxide synthase-2 in uterine arteries following stimulation with lipopolysaccharide.

Prostaglandin-endoperoxide synthase (PTGS) (also known as cyclooxygenase) converts arachidonic acid into several prostaglandins, many of which have roles in vasodilation and vasoconstriction under normal and pathological conditions. There are two isoforms of PTGS: PTGS-1 and PTGS-2; PTGS-1 is constitutively expressed in many tissues and is believed to be involved in the homeostatic maintenance of the body. In contrast, PTGS-2 is believed to have a "differentiative" role in the cells and is highly inducible during inflammation and in response to lipopolysaccharide (LPS). Endothelial cells as well as vascular smooth muscle cells can be a source of PTGS within the artery. The objective of this study was to determine the cell population(s) in uterine arteries that respond to LPS with an increase in PTGS-2 protein expression. Uterine arteries collected from ewes during the follicular (Day 0, Day 0 = estrus, n = 4) or luteal (Day 10, n = 4) phase were treated in vitro with LPS as intact artery segments, cut-open artery segments, or cut-open and denuded (endothelial cells absent) artery segments. After 24 h of LPS treatment, intact, cut-open, and denuded uterine artery segments were collected into homogenization buffer for determination of PTGS-2 protein levels by Western blot analysis. The culture medium was collected and used for detection of 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), the stable metabolite of prostacyclin, using an enzyme immunoassay. In addition, the location of PTGS-2 after LPS treatment was analyzed by immunohistochemistry in intact artery segments. Denuded arteries (endothelium absent) did not show increases in PTGS-2 protein in the homogenates or 6-keto-PGF(1alpha) in the culture medium after LPS exposure. In contrast, cut uterine arteries responded to LPS stimulation with a significant increase in PTGS-2 protein in homogenates and 6-keto-PGF(1alpha) in culture medium. Immunohistochemical staining for PTGS-2 was associated with both endothelial cells and vascular smooth muscle cells. These results suggest that while both endothelial cells and vascular smooth muscle cells are associated with PTGS-2, after LPS exposure it is the endothelial cells that are essential in uterine artery increases in PTGS-2 and prostacyclin in response to LPS stimulation.

Development and viability of bovine preplacentation embryos treated with swainsonine in vitro.

This study investigated the effects of swainsonine (a locoweed toxin) on bovine preplacentation embryo development using in vitro procedures. We examined and confirmed the viability and developmental potential of swainsonine-treated embryos by transfer to synchronized recipient heifers. Oocytes (n = 6338) were aspirated from ovaries collected from the abattoir and subjected to in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). Swainsonine was added to IVM, IVF, IVC media spatially and IVM/IVF/IVC continuously, at 0 ng/ml (TRTI, control), 200 ng/ml (TRT2), 400 ng/ml (TRT3), and 800 ng/ml (TRT4). Embryo development was evaluated with respect to oocyte cleavage rate and the rates of morula and blastocyst formation. There was no difference (P > 0.05) among treatments. The average number of nuclei per blastocyst at Day 7.5 of culture (Day 0 = IVF) was 85.9 +/- 4.3 (n = 47) and 89.3 +/- 4.4 (n = 44) for swainsonine-treated embryos (800 ng/ml) and control embryos, respectively. Pregnancy rate as determined by ultrasonography on day 35 to 40 post embryo transfer was 43.8% and 38.3% for swainsonine-treated (800 ng/ml) and control embryos, respectively. Nine (9.4%) healthy calves were delivered from heifers receiving swainsonine-exposed and nine (9.6%) from control embryos. No difference (P > 0.05) was detected in number of calves developing from TRT and control embryos. We conclude that swainsonine does not have an adverse effect on the development and viability of preplacentation bovine embryos.