The effects of follicle stimulating hormone (FSH)-induced controlled ovarian hyperstimulation and nutrition on implantation-related gene expression in caruncular tissues of non-pregnant sheep.

Disturbances at the conceptus-maternal interface can have detrimental effects on pregnancy outcome. Additionally, changes in body condition and exogenously administered gonadotropins could affect ovarian and uterine function, including cell proliferation and ovulation rates, and alter endometrial receptivity. In ruminants, endometrial caruncles maintain placental function via interaction with fetal chorionic cotyledons. Here, the effects of feeding regimens on the expression of selected genes known to be involved in uterine receptivity were investigated in the caruncles of control and FSH-superovulated ewes. Sheep were grouped according to their diet: control fed (CF), overfed (OF) or underfed (UF), and were either superovulated with FSH (SOV) or untreated (CON, naturally cycling) (n = 3-5/group). Caruncular samples for the assessment of the transcript levels of 11 target genes were collected at either the early (day 5) or mid-luteal (day 10) phases of the luteal lifespan, resulting in 12 groups of animals. The day of the estrous cycle affected the expression of ITGAV, ITGB3, FGF10 and IGFBP3 mRNA. There was lower expression of MUC1, and higher expression of FGF10, ITGB3 and FN1, on day 10 in CF_SOV animals. Compared with CF, expression of integrins (ITGB3, ITGA5 and ITGA4) was higher in OF and UF, and higher transcript levels of HGF and IGFBP3 in UF animals on day 10. Expression of ITGA5, ITGB1, -3, -5 and MUC1 was greater in OF_SOV than CF_SOV at day 10. In conclusion, it appears that imbalanced nutrition, by altering the expression of genes responsible for intercellular communication, cell adhesion, and encoding for growth factors, could affect the uterine responsiveness to exogenously applied hormonal stimulation and, likely, uterine receptivity.

Plane of nutrition and FSH-induced superovulation affect the expression of steroid hormone receptors and growth factors in caruncular tissue of non-pregnant sheep.

Implantation is a critical step in the establishment of pregnancy and an important part of embryo-maternal contact. Uterine receptivity can be affected by changes in body condition and the maternal endocrine milieu, including those caused by the use of exogenous gonadotropins in controlled ovarian hyperstimulation to induce the development of multiple follicles. This study demonstrates the effects of FSH-mediated ovarian hyperstimulation on the caruncles of ewes under various feeding regimes. Sheep were classified into 3 categories: control fed (CF), overfed (OF), or underfed (UF). In each group, animals were superovulated with FSH or injected with a saline solution (non-treated control). Uterine caruncles were collected at the early (d 5) and mid-luteal phase (d 10) of the estrous cycle. The transcript levels of steroid hormone receptors (ESR1, ESR2, PGR) and growth factors (IGF1, IGF2, VEGFA) were investigated and their expression localized by immunohistochemical staining. As for the main findings, day of the estrous cycle affected expression of ESR1, IGF1 and IGF2, but not of ESR2, PGR and VEGFA; both feeding and superovulation had modulatory effects, with feeding (UF/OF) stimulating expression of all genes studied, and superovulation altering expression of some genes, eg IGF1, PGR and ESR1 and ESR2, in CF animals. Similarly, feeding (UF/OF) altered responsiveness to superovulation for PGR on d 5 and ESR1/ESR2 on d 5 and/or 10. Our data emphasize possible effects of dietary and/or hormonal stimuli on uterine physiology, which may affect pregnancy outcomes by disrupting uterine functionality.

Mid- to late-gestational maternal nutrient restriction followed by realimentation alters development and lipid composition of liver and skeletal muscles in ovine fetuses.

Maternal nutrient restriction during gestation adversely affects offspring growth and development of liver and skeletal muscle tissues. Realimentation following nutrient restriction may alleviate these negative impacts on development but may alter metabolism and tissue composition. Forty-eight ewes, pregnant with singletons, were fed to meet 100% National Research Council (NRC) recommendations starting at the beginning of gestation. On day 50 of gestation, seven ewes were euthanized (BASE), and fetal liver, skeletal muscles, and blood samples were collected. The remaining animals were fed either 100% of NRC recommendations (CON) or 60% NRC recommendations (RES), a subset were euthanized at day 90 of gestation (n = 7/treatment), and fetal samples were collected. Remaining ewes were maintained on the current diet (CON-CON, n = 6; RES-RES, n = 7) or switched to the alternate diet (CON-RES, RES-CON; n = 7/treatment). On day 130 of gestation, the remaining ewes were euthanized, and fetal samples were collected. At day 130 of gestation, maternal nutrient restriction during late-gestation (RES-RES and CON-RES) decreased fetal liver weight (P < 0.01) and cross-sectional area in triceps brachii (P = 0.01; TB), longissimus dorsi (P = 0.02; LM), and semitendinosus (P = 0.05; STN) muscles. Maternal nutrient restriction during mid-gestation increased hepatocyte vacuole size at day 130 of gestation. Late-gestational maternal nutrient restriction increased mRNA expression of insulin-like growth factor (IGF) binding protein-1 (P < 0.01), glycogen synthase 2 (P = 0.01; GYS2), and pyruvate dehydrogenase kinase 1 (P < 0.01; PDHK1) in the liver and IGF receptor 1 (P = 0.05) in the LM. Lipid concentration in the LM was decreased by late-gestational nutrient restriction (P = 0.01) and increased by mid-gestational nutrient restriction in STN (P = 0.03) and TB (P < 0.01). Principal component analysis of lipidomics data demonstrated clustering of principal components by day of gestation and elastic net regression identified 50, 44, and 29 lipids that classified the treatments in the fetal liver, LM, and blood, respectively. In conclusion, restricting maternal nutrition impacts fetal liver and muscle morphology, gene expression, and lipid metabolism, whereas realimentation attenuated some of these effects. Therefore, realimentation may be a viable strategy to reduce the impacts of nutrient restriction, but can lead to alterations in lipid metabolism in sheep.

Maternal periconceptual nutrition, early pregnancy, and developmental outcomes in beef cattle.

The focus of this review is maternal nutrition during the periconceptual period and offspring developmental outcomes in beef cattle, with an emphasis on the first 50 d of gestation, which represents the embryonic period. Animal agriculture in general, and specifically the beef cattle industry, currently faces immense challenges. The world needs to significantly increase its output of animal food products by 2050 and beyond to meet the food security and agricultural sustainability needs of the rapidly growing human population. Consequently, efficient and sustainable approaches to livestock production are essential. Maternal nutritional status is a major factor that leads to developmental programming of offspring outcomes. Developmental programming refers to the influence of pre-and postnatal factors, such as inappropriate maternal nutrition, that affect growth and development and result in long-term consequences for health and productivity of the offspring. In this review, we discuss recent studies in which we and others have addressed the questions, "Is development programmed periconceptually?" and, if so, "Does it matter practically to the offspring in production settings?" The reviewed studies have demonstrated that the periconceptual period is important not only for pregnancy establishment but also may be a critical period during which fetal, placental, and potentially postnatal development and function are programmed. The evidence for fetal and placental programming during the periconceptual period is strong and implies that research efforts to mitigate the negative and foster the positive benefits of developmental programming need to include robust investigative efforts during the periconceptual period to better understand the implications for life-long health and productivity.

Nuclear and membrane progesterone receptors expression in placenta from early to late pregnancy in sheep: Effects of restricted nutrition and realimentation.

Nutrient restriction and/or realimentation may affect several placental functions, such as expression of selected regulatory factors, blood flow and other processes in sheep and other species. To determine the effects of the plane of nutrition, nulliparous white face ewes (6-8 months) carrying singletons on day 50 of gestation were randomly assigned to two dietary treatments receiving 100% of National Research Council recommendations (control; C) or 60% of C (restricted; R). Two groups remained on C or R diets from day 50 until day 130. From day 90-130 another group of C fed ewes was switched to the R diet, and another group of R fed ewes was switched to the C diet. This resulted in 7 groups (n = 5-6 ewes/group): C (day 50, 90 and 130), R (day 90 and 130), CR (day 130) and RC (day 130). At these time points, placental tissues were collected for the evaluation of progesterone receptor (PGR) protein expression (whole tissue), and mRNA expression in maternal (caruncular, CAR) and fetal (cotyledon, COT) (separated tissues). Data were statistically analyzed using analysis of variance (SAS 9.4). Protein for PGRAB and PGRB isoforms was detected using immunohistochemistry in all placental tissues, but the pattern of expression differed depending on pregnancy stage and placental compartment (e.g., CAR vs COT). PGRAB protein expression, quantified using image analysis, was greater (P < 0.04) on day 50 than 90 or 130, and was not affected by plane of nutrition. In CAR and COT, PGRAB mRNA expression was greater (P < 0.05) on day 50 than 90 or 130. PGRB mRNA expression was greater (P < 0.03) in CAR on day 50 than 90 and 130, and was greatest (P < 0.02) in COT on day 50, less on day 130, and least on day 90. For the membrane progesterone receptors, PAQR7 (membrane PGR alpha) mRNA expression was greater (P < 0.05) on days 50 and 90 than 130 in CAR, and greater (P < 0.01) on days 50 than 90 and 130 in COT; PAQR8 (membrane PGR beta) was similar throughout pregnancy in CAR and COT, and PAQR5 (membrane PGR gamma) was greatest (P < 0.0001) on day 130 in COT, but similar throughout pregnancy in CAR. Plane of nutrition affected (P < 0.05) mRNA expression for all genes in CAR and COT throughout pregnancy. These data indicate that expression of PGR in ovine placenta is dependent on stage of pregnancy and plane of nutrition in sheep. The mechanisms of how diet and stage of pregnancy influences placental PGR expression and function remains to be elucidated.

Lipopolysaccharide disrupts gap junctional intercellular communication in an immortalized ovine luteal endothelial cell line.

Gram-negative bacteria, in particular Escherichia coli with its cell wall lipopolysaccharide (LPS), often cause metritis and mastitis in domestic animals. Ovarian LPS accumulation may initiate local inflammatory reactions mediated through cell surface Toll-like receptors (TLRs). This may disrupt ovarian functionality leading to infertility. Possible adverse effects of LPS on luteal activity are not yet well explored. We hypothesized that LPS could lead to alterations in luteal vascular functionality. Therefore, we established an in vitro cell line model (OLENDO) by immortalizing microvascular endothelial cells isolated from ovine corpus luteum (CL) with a potent Simian Virus 40 T-antigen (SV40-Tag). OLENDO exhibit endothelial cell characteristics, like low-density lipoprotein (LDL) uptake, express BSL-I, and VEGFR2, as well as TLR2 and TLR4 receptors. LPS-treatment of OLENDO altered in vitro tube formation, had no effects on cell viability and decreased gap junctional intercellular communication (GJIC). LPS did not impair GJA1/Cx43 protein expression, but altered its cellular localization showing signs of internalization. Taken together, we demonstrated the mechanisms underlying LPS induced impairment of luteal GJIC and immune processes in a novel and well-characterized OLENDO cell line.

Placental development during early pregnancy in sheep: nuclear estrogen and progesterone receptor mRNA expression in the utero-placental compartments.

Sex steroid hormones are major regulators of uterine and placental growth and functions, as well as many other biological processes. To examine the mRNA expression of nuclear estrogen (ESR1 and 2) and progesterone (PGRAB and B) receptors in different compartments of the uterus and placenta, tissues were collected in experiment 1 on days 16, 20, and 28 after natural mating (NAT) and on day 10 after estrus (nonpregnant controls [NP]); and in experiment 2 on day 22 of NAT, and pregnancies established after transfer of embryos generated through mating of FSH-treated ewes (NAT-ET), in vitro fertilization (IVF), or in vitro activation (parthenotes). In experiment 1, ESR1 expression in endometrial stroma (ES), endometrial glands (EGs), and myometrial blood vessels (MBVs), ESR2 in endometrial blood vessels (EBV), PGRAB in ES, and PGRB in ES, EG, and MBV was greater in pregnant than NP ewes depending on the day of pregnancy. The day of pregnancy affected the expression of ESR1 in MBV, ESR2 in EBV and MBV, and PGRAB in ES. In experiment 2, ESR1, PGRAB, and PGRB in EG, but not in other compartments, was greater in NAT-ET than NAT, and PGRB was greater for NAT-ET than IVF. These data demonstrate that ESR and PGR expression differ in pregnant versus NP ewes in selected compartments and was affected by pregnancy stage or embryo origin in selected utero-placental compartments. Thus, sex steroid hormone mRNA expression is differentially regulated in a spatiotemporal manner in the uterus and placenta and is affected by the application of assisted reproductive technology in sheep.

Effects of plane of nutrition and arginine on ovarian follicles in non-pregnant sheep: Cell proliferation, and expression of endothelial nitric oxide and its receptor.

The aim of this study was to investigate the role of the nitric oxide (NO) system in ovarian function, by determining if arginine (Arg) supplementation impacts follicle number, cell proliferation, and expression of the NO system members in nutritionally compromised ewes. Ewes were randomly assigned into maintenance (C, 100% requirements), excess (O; 2xC), or restricted (U; 0.6xC) diets 8 weeks prior to Arg treatment. Ewes were individually fed twice daily with pelleted diets. Ewes from each nutritional group were randomly assigned to one of two treatments: saline or Arg, which was initiated on day 0 of the estrous cycle and administered 3 times per day. Ovaries were collected at the early-luteal, mid-luteal and late-luteal/follicular phases of the estrous cycle to determine 1) the number of surface follicles, 2) follicle cell proliferation marked by Ki67 protein expression, and 3) expression of endothelial nitric oxide (eNOS; NOS3) and soluble guanylyl cyclase beta (sGC; GUCY1B3) protein and mRNA in granulosa (G) and theca (T) layers using immunohistochemistry followed by image analysis and qPCR, respectively. During nutritional treatment, C maintained body weight, O gained 6±1.2 kg, and U lost 14±1.3 kg. Our data show that: 1) Ki67 was expressed in all ovarian compartments, eNOS protein was detected in blood vessels of T and stroma, and sGC protein was detected in T cells, and blood vessels of T layer and other ovarian compartments; 2) plane of nutrition affected the number of surface follicles, and thus folliculogenesis, cell proliferation in the T layer, eNOS and sGC protein expression in T, and NOS3 and GUCY1B3 mRNA expression in G; 3) Arg treatment affected cell proliferation in G and T, eNOS and sGC protein expression in T, mRNA expression of NOS3 in T in all groups, and GUCY1B3 in G depending on the stage of the estrous cycle; and 4) G and T cell proliferation, and expression of eNOS and sGC protein in T was affected by the stage of the estrous cycle. Our data demonstrated that plane of nutrition and Arg are involved in the regulation of follicular functions in non-pregnant sheep.

Angiopoietin expression in ovine corpora lutea during the luteal phase: Effects of nutrition, arginine and follicle stimulating hormone.

The aim of this study was to evaluate angiopoietin (ANGPT) 1 and 2, and tyrosine-protein kinase receptor 2 (TIE2) expression in the corpora lutea (CL) of FSH-treated, or non-treated sheep administered arginine (Arg) or vehicle (saline, Sal), and fed a control (C), excess (O) or restricted (U) diet. Ewes from each dietary group were treated with Arg or Sal (experiment 1), and with FSH (experiment 2). Luteal tissues were collected at the early-, mid- and/or late-luteal phases of the estrous cycle. Protein and mRNA expression was determined using immunohistochemistry followed by image analysis, and quantitative RT-PCR, respectively. The results demonstrated that ANGPT1 and TIE2 proteins were localized to luteal capillaries and endothelial cells of larger blood vessels, and ANGPT2 was localized to tunica media of larger blood vessels. TIE2 protein was also present in luteal cells. In experiment 1, ANGPT1 protein expression was greater in O than C during early- and mid-luteal phases, and was greatest during late-luteal phase, less at the mid- and least at the early-luteal phase; 2) TIE2 protein expression was greatest at the mid-, less at the early- and least at the late-luteal phase; 3) ANGPT1 and 2 mRNA expression was greater at the mid- and late- than the early-luteal phase, and TIE2 mRNA expression was greatest at the late-, less at the mid- and least at the early-luteal phase. The ANGPT1/2 ratio was less at the early- than mid- or late-luteal phases. In experiment 2, ANGPT1 protein expression was greater in O during the mid-luteal phase than in other groups, and was greater at the mid- than early-luteal phase. TIE2 protein expression was highest at the mid-, less at the early- and least during the late-luteal phase. ANGPT1 and 2, and TIE2 mRNA expression was higher at the mid- than the early-luteal phase. During mid-luteal phase, ANGPT1 mRNA expression was greater in C than O and U, ANGPT2 was greatest in C, less in O and least in U, and TIE2 mRNA expression was greater in C than O and U. The ANGPT1/2 ratio was higher in U than in any other group. Comparison of FSH vs. Sal treatment effects (experiment 2 vs. experiment 1) demonstrated that FSH affected ANGPT1 and/or -2, and TIE2 protein and mRNA expression depending on luteal phase and/or diet. Thus, expression of ANGPTs and TIE2 in the CL changes during the luteal lifespan, indicating their involvement in luteal vascular formation, stabilization and degradation. Moreover, this study has demonstrated that plane of nutrition and/or FSH treatment affect the ANGPT system, and may alter luteal vascularity and luteal function in sheep.

Luteal ANGPT-TIE system during selected stages of pregnancy, and normal and antigestagen-induced luteolysis in the dog.

Abstract: Rapid establishment of a vascular network is essential for normal functionality of the corpus luteum (CL). The early luteal phase is associated with increased expression of the VEGF system in canine CL. Acting in synchrony with angiopoietins (ANGPTs), VEGF system plays major roles in stabilization of blood vessels. However, the expression of the ANGPT system has not yet been investigated in the dog. Therefore, here, we investigated the luteal expression of ANGPT1, -2, and of their receptors TIE1 and -2, in pregnant dogs at selected time points during pregnancy and at normal and antigestagen-induced luteolysis. Additionally, luteal cells from early CL were incubated with PGE2 and its effects on the ANGPT system were assessed. Whereas the luteal ANGPT1 was stable until mid-gestation, TIE1 was elevated post-implantation, their expression decreased toward prepartum luteolysis. The ANGPT2- and TIE2-mRNA did not vary during pregnancy. The ANGPT2/ANGPT1 ratio was elevated during prepartum luteolysis. PGE2 increased ANGPT2, but suppressed ANGPT1 levels. None of the ANGPT-system members was affected by antigestagen treatment in mid-pregnancy. Localization of ANGPT1 was predominantly found in the tunica intima and media of vessels and ANGPT2 stained strongly in luteal cells. Both ANGPTs were localized in macrophages. TIE1 stained in the vascular tunica media, in luteal cells and macrophages, whereas TIE2 was colocalized with ANGPT1 in vascular components. In conclusion, high expression of ANGPT1 during the increased presence of VEGFA in early canine CL implies its contribution to vascular network development. The upregulation of the ANGPT2/ANGPT1 ratio during prepartum luteolysis indicates involvement of the ANGPT system in PGF2α-mediated vascular destabilization.

Follicle stimulating hormone receptor protein is expressed in ovine uterus during the estrous cycle and utero-placenta during early pregnancy: An immunohistochemical study.

Follicle stimulating hormone (FSH) is a well characterized gonadotropin that controls primarily development and functions of ovarian follicles in mammalian species. FSH binds to a specific G protein-coupled receptor (FSHR) belonging to the glycoprotein hormone receptor family that plays an essential role in reproduction. Although the primary location of FSHR is in the gonads (mainly in ovarian follicles), FSHR protein and/or mRNA have also been detected in extragonadal female reproductive tissues including embryo, placenta, endometrium, cervix, ovarian cancer tissues, and/or endometriotic lesions in several species. To determine the pattern of FSHR expression in the uterus and placenta, uterine tissues were collected at the early, mid- and/or late luteal phases of the estrous cycle from non-treated or FSH-treated ewes, and utero-placental tissues were collected during early pregnancy followed by immunohistochemistry and image generation. FSHR was immunolocalized to several uterine and utero-placental compartments including luminal epithelium, endometrial glands and surrounding stroma, myometrium, and endothelium and vascular smooth muscle cells in endometrium, myometrium and mesometrium. Intensity of staining and distribution of FSHR in selected compartments differed and seems to depend on the stage of the estrous cycle or pregnancy, and FSH-treatment. These novel data demonstrate differential expression of FSHR protein indicating that FSH plays a specific role in regulation of uterine and utero-placenta functions in sheep.

Placental development during early pregnancy in sheep: Progesterone and estrogen receptor protein expression.

The aim of this study was to evaluate the pattern of protein expression of the steroid receptor isoforms of nuclear progesterone receptors (PGR) A and B, and estrogen receptors (ESR1 and 2) in utero-placental compartments during early pregnancy. Utero-placental tissues were collected from days 14-30 (n = 4 ewes/day), and uterine tissues were collected from non-pregnant ewes on day 10 after estrus (n = 4). Cross sections of formalin-fixed and paraffin embedded tissues were immunofluorescently stained to detect PGRAB, PGRB, ESR1 and ESR2, followed by image generation of entire cross-sections of uterine and utero-placental tissues, confocal imaging of individual uterine and utero-placental compartments, and image and statistical analyses. PGRAB, PGRB, ESR1 and ESR2 were detected in several compartments of uterine and utero-placental tissues. Quantitative image analysis of staining intensity demonstrated that compared to non-pregnant controls 1) expression of PGRAB and PGRB was less in luminal epithelium and endometrial glands from day 14-16 till 30; 2) PGRAB expression tended to be greater in endometrial and myometrial blood vessels on days 28 and/or 30; 3) PGRB expression in myometrum was lower on days 16 and 28; 4) ESR1 in endometrial stroma was lower in all days of pregnancy; 5) ESR2 expression was similar in all compartments and not affected by pregnancy stage; and 6) in FM, expression of steroid receptors was similar. Thus, we have demonstrated spatial and temporal expression of nuclear PGR and ESR isoforms in utero-placental compartments during early pregnancy.

Expression of progesterone receptor protein in the ovine uterus during the estrous cycle: Effects of nutrition, arginine and FSH.

To evaluate expression of progesterone receptor (PGR) AB in follicle stimulating hormone (FSH)-treated or non-treated sheep administered with arginine (Arg) or saline (Sal) fed a control (C), excess (O) or restricted (U) diet, uterine tissues were collected at the early, mid and/or late luteal phases. In exp. 1, ewes from each diet were randomly assigned to one of two treatments, Arg or Sal administration three times daily from day 0 of the first estrous cycle until uterine tissue collection. In exp. 2, ewes were injected twice daily with FSH on days 13-15 of the first estrous cycle. Uterine tissues were immunostained to detect PGR followed by image analysis. PGR were detected in luminal epithelium (LE), endometrial glands (EG), endometrial stroma (ES), myometrium (Myo), and endometrial and myometrial blood vessels. The percentage of PR-positive cells and/or intensity of staining were affected by phase of the estrous cycle, plane of nutrition, and/or FSH but not by Arg. In exp. 1, percentage of PGR-positive cells in LE and EG but not in ES and Myo was greater at the early and mid than late luteal phase, was not affected by plane of nutrition, and was similar in LE and EG. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in LE, EG and Myo, and was the greatest in LE, less in EG, and least in ES and Myo. In exp. 2, percentage of PGR-positive cells in LE, EG, ES and Myo was affected by phase of the estrous cycle, but not by plane of nutrition; was greater at the early than mid luteal phase; and was greatest in LE and EG, less in luminal (superficial) ES and Myo and least in deep ES. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in all compartments but ES, and was the greatest in LE and luminal EG, less in deep EG, and least in ES and Myo. Comparison of data for FSH (superovulated) and Sal-treated (non-superovulated) ewes demonstrated that FSH affected PR expression in all evaluated uterine compartments depending on plane of nutrition and phase of the estrous cycle. Thus, PGR are differentially distributed in uterine compartments, and PGR expression is affected by nutritional plane and FSH, but not Arg depending on phase of the estrous cycle. Such changes in dynamics of PGR expression indicate that diet plays a regulatory role and that FSH-treatment may alter uterine functions.

A Preliminary Evaluation of Ovine Bladder Mucosal Damage Associated With 2 Different Indwelling Urinary Catheters.

OBJECTIVE: To determine whether a new catheter design with a low-profile, open-ended rounded rather than pointed Foley catheter tip can reduce mucosal damage to the bladder of ewes. METHODS AND MATERIALS: Six ewes were randomly assigned to 1 of 2 indwelling urinary catheters-a 16 Fr Foley catheter or a 16 Fr open-tip CystoSure catheter. After 96 hours, all the animals were sacrificed and their bladder and urethra harvested for analysis. RESULTS: Image analysis of the bladder surfaces demonstrated a significant decrease in the percentage of bladder area covered by ulceration and inflammation in sheep with CystoSure catheters compared with Foley catheters (P < .002) as well as a trend toward less edema (P = .17). Macro-morphologic evaluations were confirmed with immunohistochemical markers of cell proliferation and inflammation. CONCLUSION: In this pilot study, we were able to demonstrate that a new catheter design with an open-ended rounded rather than pointed tip and a reduced balloon base-to-tip profile may reduce mucosal damage to the bladder of ewes. Based on the findings from this trial, we believe this new catheter design with its low-profile, rounded tip may reduce bladder mucosal injury, which is a risk factor for catheter-associated urinary tract infections.

Luteal function during the estrous cycle in arginine-treated ewes fed different planes of nutrition.

Functions of corpus luteum (CL) are influenced by numerous factors including hormones, growth and angiogenic factors, nutritional plane and dietary supplements such as arginine (Arg), a semi-essential amino acid and precursor for proteins, polyamines and nitric oxide (NO). The aim of this study was to determine if Arg supplementation to ewes fed different planes of nutrition influences: (1) progesterone (P4) concentrations in serum and luteal tissue, (2) luteal vascularity, cell proliferation, endothelial NO synthase (eNOS) and receptor (R) soluble guanylate cyclase ß protein and mRNA expression and (3) luteal mRNA expression for selected angiogenic factors during the estrous cycle. Ewes (n = 111) were categorized by weight and randomly assigned to one of three nutritional planes: maintenance control (C), overfed (2× C) and underfed (0.6× C) beginning 60 days prior to onset of estrus. After estrus synchronization, ewes from each nutritional plane were assigned randomly to one of two treatments: Arg or saline. Serum and CL were collected at the early, mid and late luteal phases. The results demonstrated that: (1) nutritional plane affected ovulation rates, luteal vascularity, cell proliferation and NOS3, GUCY1B3, vascular endothelial growth factor (VEGF) and VEGFR2 mRNA expression, (2) Arg affected luteal vascularity, cell proliferation and NOS3, GUCY1B3, VEGF and VEGFR2 mRNA expression and (3) luteal vascularity, cell proliferation and the VEGF and NO systems depend on the stage of the estrous cycle. These data indicate that plane of nutrition and/or Arg supplementation can alter vascularization and expression of selected angiogenic factors in luteal tissue during the estrous cycle in sheep.

Lipid droplets in the ovine uterus during the estrous cycle: Effects of nutrition, arginine, and FSH.

The aim of this study was to evaluate lipid droplet (LD) expression in uteri of FSH-treated or nontreated sheep administered with arginine (Arg) or vehicle (saline, Sal) and fed a control (C), excess (overfed, O) or restricted (underfed, U) diet. In experiment 1, ewes from each diet were randomly assigned to Arg or Sal treatments administered three times daily starting on Day 0 of the first estrous cycle until blood sample and uterine tissue collection at the early- or mid-luteal phase of the second estrous cycle or the late-luteal phase of the first estrous cycle. In experiment 2, ewes were injected twice daily with FSH on Days 13 to 15 of the first estrous cycle, and blood samples and uterine tissue were collected at the early- and mid-luteal phases of the second estrous cycle. Cryopreserved in optimum cutting temperature (OCT) compound, cross-sections of uterine horn were stained with boron-dipyrromethene (BODIPY; marker of LDs) followed by 4',6-diamidino-2-phenylindole (DAPI) staining and image analysis to determine the proportion (%) of area occupied by LD in luminal epithelium (LE) and endometrial glands (EGs). Control ewes maintained, O ewes gained, and U ewes lost body weight during the experiments. Serum progesterone concentration was not affected by nutritional plane or Arg treatment and was 5.5-fold greater in FSH- than Sal-treated ewes. LDs were detected in LE and superficial EG (close to LE) but not deep EG, or other uterine compartments, and area occupied by LD was greater in LE than in EG. In experiment 1, in LE and EG, area occupied by LDs was greater in C than in O or U; greater in Arg than in Sal; and greater at the late-, less at mid-, and least at early-luteal phases. In experiment 2, in LE and EG, area occupied by LDs was greater at mid- than in early-luteal phase. Comparison of data from FSH-treated and nontreated ewes (e.g., experiment 1 vs. experiment 2) demonstrated that FSH increased the area occupied by LD in LE and EG regardless of diet. Interactions between FSH treatment, stage of the estrous cycle, and plane of nutrition demonstrated that FSH increased the area occupied by LD in LE and EG at the mid-luteal phase in O and U. Thus, LDs are differentially distributed in uterine compartments, and area occupied by LD in endometrium is affected by nutritional plane, Arg or FSH, and stage of the estrous cycle. Such changes in dynamics of LD in the endometrium during the estrous cycle indicate their specific role in uterine functions.

Lipid droplets in cultured luteal cells in non-pregnant sheep fed different planes of nutrition.

Accumulation of lipid droplets (LD) in luteal cells likely is important for energy storage and steroidogenesis in the highly metabolically active corpus luteum (CL). The objective of this study was to determine the effect of plane of nutrition on progesterone (P4) secretion, and lipid droplet number and size in cultured ovine luteal cells. Ewes were randomly assigned to one of three nutritional groups: control (C; 100% NRC requirements, n=9), overfed (O; 2×C, n=12), or underfed (U; 0.6×C, n=10). Superovulation was induced by follicle stimulating hormone injections. At the early and mid-luteal phases of the estrous cycle, CL were dissected from ovaries, and luteal cells isolated enzymatically. Luteal cells were incubated overnight in medium containing serum in chamber slides. Media were then changed to serum-free and after 24h incubation, media were collected for P4 analysis, and cells were fixed in formalin and stained with BODIPY followed by DAPI staining. Z-stacks of optical sections of large and small luteal cells (LLC and SLC, respectively) were obtained using a laser-scanning microscope. Rendered 3D images of individual LLC and SLC were analyzed for cell volume, and total and individual LD volume, number and percentage of cellular volume occupied by LD by using Imaris software. Concentrations of P4 in serum and media were greater (P<0.05) at the mid than early-luteal phase, and were not affected by nutritional plane. LD total volume and number were greater (P<0.001) in LLC than SLC; however, mean volume of individual LD was greater (P<0.02) in SLC than LLC. In LLC, total LD volume was greater (P<0.02) in O than C and U ewes. In SLC, total LD volume and number was greater (P<0.003) at the mid than early-luteal phase, and percentage of cell volume occupied by LD was greater (P<0.002) in U than C and O ewes. These data demonstrate that both stage of luteal development and nutritional plane affect selected LD measurements and thus may affect luteal functions. Furthermore, these data confirm that LD dynamics differ among parenchymal steroidogenic luteal cell types.

Progesterone secretion by ovine granulosa cells: effects of nitric oxide and plane of nutrition.

The aim was to evaluate the effects of nutritional plane on in vitro progesterone (P4) secretion by granulosa (G) cells cultured in the presence or absence of effectors of the nitric oxide (NO) system. Ewes were randomly assigned into three nutritional groups: control (C), overfed (O; 2 × C), or underfed (U; 0.6 × C). Follicular development was induced by FSH injections. On day 15 of the estrous cycle, G cells were isolated and cultured with or without DETA-NONOate (NO donor), L-NAME (NO synthase [S] inhibitor), Arg and (or) LH for 8 h. DETA-NONOate decreased basal and LH-stimulated P4 secretion, and L-NAME increased basal P4 secretion in all groups. In U, Arg decreased LH-stimulated P4 secretion. These data demonstrate that (i) plane of nutrition affects basal P4 secretion by G cells, (ii) the NO donor decreases, NOS inhibitor increases but Arg does not affect basal P4 secretion, and (iii) effects of Arg on LH-stimulated P4 secretion are affected by plane of nutrition in FSH-treated sheep. Thus, plane of nutrition affects G cell function, and the NO system is involved in the regulation of basal and LH-stimulated P4 secretion. The mechanism of the NO system effects on secretory activity of G cells remains to be elucidated.

The effects of diet and arginine treatment on serum metabolites and selected hormones during the estrous cycle in sheep.

The aim of this study was to determine the effects of diet and arginine (Arg) treatment on serum concentrations of selected metabolites and metabolic and reproductive hormones in nonpregnant ewes. Sixty days before the onset of estrus (Day 0), Rambouillet ewes were randomly assigned to one of three dietary groups: maintenance control (C; N = 16; 100% National Research Council requirements), overfed (O; N = 16; 2 × C), or underfed (U; N = 16, 0.6 × C) to achieve and maintain three different body conditions during their estrous cycle(s). At Day 0, ewes from each nutritional group were randomly assigned to receive one of two treatments: saline (Sal) or Arg (L-Arg-HCl; 155 µmol Arg per kg of body weight [BW]; intravenous), which was administered three times per day for 21 or 26 days. Blood samples were collected on Days 0, 6, 10, 12, 16, 21, and 26 of Sal or Arg treatment for evaluation of Arg, nitric oxide metabolite, cholesterol, glucose, insulin, insulin-like growth factor 1, leptin, and progesterone. For a time-response trial, blood samples were collected at 0, 1, 2, 4, and 7 hours after Sal or Arg treatment at the mid-luteal phase to determine serum Arg concentrations. During the 11-week study, C maintained body weight, O gained 9.6 ± 0.7 kg, and U lost 13.9 ± 0.1 kg. Overall, serum concentrations of Arg, glucose, insulin, insulin-like growth factor 1, leptin, and progesterone were greater (P < 0.05) in O ewes than C and/or U ewes and were not affected by Arg treatment. Serum Arg concentration increased at 1 and 2 hours and decreased to basal level at 4 and 7 hours after Arg treatment. These data reinforce the importance of diet in regulation of metabolic and endocrine functions, and demonstrated that the dose and duration of Arg treatment used in this study does not alter serum metabolites or hormones in nonpregnant ewes of various nutritional planes.

Placental vascular defects in compromised pregnancies: effects of assisted reproductive technologies and other maternal stressors.

Many factors negatively affect pregnancy establishment and subsequent fetal growth and development, including maternal factors such as nutritional stress, age, body mass index, and genetic background, and external factors including environmental stress, psychosocial stress, multiple fetuses, medical conditions (e.g., polycystic ovary syndrome), lifestyle choices (e.g., alcohol consumption, smoking), and assisted reproductive technologies. These same factors have similar consequences for placental growth and development, including vascular development. We and others have shown that placental vascular development begins very early in pregnancy and determines, to a large extent, placental function-that is, the magnitude of the increase in placental blood flow and thus nutrient transport to the fetus. During the peri-implantation period and also later in pregnancy, cloned (somatic cell nuclear transfer) embryos exhibit a variety of placental defects including reduced vascularization and altered expression of angiogenic factors. Although placental defects are less pronounced in pregnancies resulting from the transfer of in vitro fertilized embryos, we and others have recently demonstrated that vascularization, expression of angiogenic factors, sex steroid receptors, several epigenetic markers, and growth of utero-placental tissues all were altered during early pregnancy after transfer of embryos obtained through natural mating, in vitro fertilization, or other assisted reproductive techniques. These observations are in agreement with the recent reports that in humans even singleton pregnancies established with assisted reproductive techniques are at increased risk of preterm delivery and low birth weight, and seem especially relevant considering the rapidly expanding use of these techniques in humans and animals.