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.

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.

Effect of melatonin or maternal nutrient restriction on vascularity and cell proliferation in the ovine placenta.

Previously we reported increased umbilical artery blood flow in ewes supplemented with melatonin from mid- to late-pregnancy, while maternal nutrient restriction decreased uterine artery blood flow. To further unravel these responses, this study was designed to assess placental cell proliferation and vascularity following supplementation with melatonin or maternal nutrient restriction. For the first experiment, 31 primiparous ewes were supplemented with 5mg of melatonin per day (MEL) or no melatonin (CON) and allocated to receive 100% (adequate fed; ADQ) or 60% (restricted; RES) of their nutrient requirements from day 50 to 130 of gestation. To examine melatonin receptor dependent effects, a second experiment was designed utilizing 14 primiparous ewes infused with vehicle, melatonin, or luzindole (melatonin receptor 1 and 2 antagonist) from day 62 to 90 of gestation. For experiment 1, caruncle concentrations of RNA were increased in MEL-RES compared to CON-RES. Caruncle capillary area density and average capillary cross-sectional area were decreased in MEL-RES compared to CON-RES. Cotyledon vascularity was not different across dietary treatments. For experiment 2, placental cellular proliferation and vascularity were not affected by infusion treatment. In summary, melatonin interacted with nutrient restriction to alter caruncle vascularity and RNA concentrations during late pregnancy. Although melatonin receptor antagonism alters feto-placental blood flow, these receptor dependent responses were not observed in placental vascularity. Moreover, placental vascularity measures do not fully explain the alterations in uteroplacental blood flow.

Ovarian and uterine characteristics and onset of puberty in adolescent offspring: effects of maternal diet and selenium supplementation in sheep.

The aim of this study was to determine the effects of maternal diet with adequate (A) or high (H) selenium (Se) supplementation on ovarian and uterine characteristics, and onset of puberty in adolescent offspring. Sheep were fed a maintenance (M) diet with ASe or HSe levels from breeding to parturition. From Day 50 to parturition, a portion of the ewes from ASe and HSe groups was fed restricted (R, 60% of M) or excess (E, 140% of M) diet. Immediately after birth, lambs were separated from their dams and given artificial colostrum for 20 hours, followed by milk replacer. From Day 57.3 ± 0.6, ewe lambs were fed a pelleted grower diet until Day 116.3 ± 0.6 when they were transitioned to a finisher diet. From Day 99 to 180, serum samples were collected weekly from jugular vein for progesterone analysis to determine onset of puberty. Reproductive tissues were collected on Day 180.1 ± 0.4 of age. Maternal diet or Se supplementation did not affect uterine or ovarian weight and onset of puberty. However, area under the curve for progesterone was greater (P = 0.05) in ASe compared with HSe groups, and was greater in ASeM than HSeM group. In CLs, labeling index (LI; a proportion of proliferating cells) was less (P < 0.04) in HSeM than ASeM group, and in stroma was less (P < 0.05) in R and E groups than M group. Maternal diet did not affect the LI of any follicle types. For all groups combined, LI was the greatest (P < 0.001) in antral, less in early antral and secondary, and the least in atretic follicles. Our results demonstrate that maternal diet influenced ovarian but not uterine characteristics or onset of puberty. These results indicate that maternal plane of nutrition and/or Se supplementation may have specific effects on reproductive function in offspring.

Thyroid Hormones and Cortisol Concentrations in Offspring are Influenced by Maternal Supranutritional Selenium and Nutritional Plane in Sheep.

To determine the effects of maternal supranutritional selenium (Se) supplementation and maternal nutritional plane on offspring growth potential, ewes were randomly assigned to 1 of 6 treatments in a 2 × 3 factorial arrangement [dietary Se (adequate Se; 9.5 µg/kg body weight vs. high Se; 81.8 µg/kg body weight initiated at breeding) and plane of nutrition [60%, 100%, or 140% of requirements; initiated on day 50 of gestation]]. Lambs were immediately removed from dams at birth and reared. Cortisol concentrations at birth were similar, but by 24 h, a relationship (P = 0.02) between maternal Se supplementation and nutritional plane on cortisol concentrations was observed in lambs. A sex of offspring × day of age interaction (P = 0.01) and a maternal Se supplementation × nutritional plane × day of age interaction (P = 0.04) was observed for thyroxine concentrations. Differences in growth may be influenced by thyroid hormone production early in neonatal life.

Neonatal hormone changes and growth in lambs born to dams receiving differing nutritional intakes and selenium supplementation during gestation.

To investigate the effects of maternal selenium (Se) supplementation and nutritional intake during gestation on hormone changes, percentage body weight (BW) change, and organ mass in neonatal lambs, ewes were allocated to differing Se levels (adequate Se (ASe, 11.5 µg/kg BW) or high Se (HSe, 77.0 µg/kg BW)) initiated at breeding and nutritional intake (60% (RES), 100% (CON), or 140% (HIGH) of NRC requirements) initiated at day 40 of gestation. At parturition, all lambs were removed from dams, fed common diets, and BW and blood samples were collected until day 19. There was a Se × nutritional intake × day interaction for percentage BW change from birth. Lambs born to ASe-HIGH ewes tended to have decreased BW change compared with ASe-CON and ASe-RES groups on day 7. Lambs from HSe-HIGH ewes tended to have increased BW change compared with HSe-RES and HSe-CON groups from days 7 to 19. At birth, there was a Se × sex of offspring interaction, in which male lambs from HSe ewes had decreased cortisol concentrations compared with all other lambs. By 24 h, lambs from RES ewes had decreased cortisol compared with those from HIGH ewes, with lambs from CON ewes being intermediate. Lambs from RES- and CON-fed ewes had greater thyroxine than HIGH ewes at 24 h. Organ masses on day 19 were mainly impacted by maternal nutritional intake and sex of the offspring. Birth weight alone did not predict growth performance during neonatal life. Moreover, despite a similar postnatal diet, maternal nutritional plane and Se status did impact neonatal endocrine profiles. Exact mechanisms of how neonatal endocrine status can influence later growth and development need to be determined.

Cellular proliferation and vascularization in ovine fetal ovaries: effects of undernutrition and selenium in maternal diet.

Sheep were fed a maintenance (M) diet with adequate (A) Se or high (H) Se concentration from 21 days before breeding to day 135 of pregnancy. From day 50 to day 135 of pregnancy (tissue collection day), a portion of the ewes from ASe and HSe groups were fed restricted (R; 60% of M) diet. Fetal ovarian sections were stained for: 1) the presence of proliferating cell nuclear antigen (a marker of proliferating cells) to determine the proportion of proliferating primordial follicles, or the labeling index (LI; percentage of proliferating cells) for primordial, primary, secondary and antral follicles, stromal tissues, and blood vessels; 2) factor VIII (a marker of endothelial cells) or 3) a presence of apoptotic cells/bodies. The number of proliferating primordial follicles and the LI of primordial follicles was decreased by R and/or HSe diets. The LI was similar for theca and granulosa cells, and for secondary or antral follicles, but was greater in secondary and antral than in primordial and primary follicles. R diet and/or Se affected the LI in all follicle types, in stromal tissues and blood vessels. A dense network of blood vessels was detected in the areas containing secondary to antral follicles, medulla, and hilus, but areas containing primordial follicles were poorly vascularized. The number of apoptotic cells was minimal. These results demonstrate that nutrient restriction and/or Se level in the maternal diet affected cellular proliferation in follicles, blood vessels, and stromal tissues in fetal ovaries. Thus, plane of nutrition and Se in the maternal diet may impact fetal ovarian development and function.

Effect of diet composition on pregnancy outcome in overnourished rapidly growing adolescent sheep.

When pregnant adolescent sheep are overnourished to promote maternal growth during pregnancy, growth of the placenta is impaired and results in the premature delivery of low birth weight lambs relative to control-fed adolescents of equivalent age. These effects have been achieved by feeding two levels of the same complete diet. The present study evaluated the role of protein in pregnancy outcome in our adolescent sheep paradigm. Adolescent ewes were implanted with single embryos on day 4 post-oestrus. Thereafter ewes were offered ad libitum an isoenergetic diet (11.4 MJ metabolisable energy/kg DM) containing either 12% (basic, B) or 17% (extra, E) crude protein. At day 75 of gestation, half the pregnant ewes on each protein level were switched to yield four groups, BB, EE, BE and EB protein. A further optimally nourished control group received a moderate quantity of a ration (14% crude protein) designed to provide 100% of the estimated energy and protein requirement of the adolescent sheep according to stage of pregnancy. Pregnancy outcome was determined at term. Feed intakes were independent of protein level in the four groups of ewes fed ad libitum and were higher (P<0.001) than in the control group throughout. Maternal plasma urea concentrations reflected the current crude protein content of the diet offered and were elevated in the 17% compared with 12% protein groups (P<0.001). Within groups fed ad libitum, maternal plasma insulin, glucose, NEFA and homocysteine concentrations were largely independent of protein level. Gestation length, placental weight, lamb birth weight and initial colostrum yield were reduced (P<0.05) in all groups fed ad libitum relative to the optimally nourished control group. Similarly, total colostrum IgG, butterfat, lactose and crude protein content at parturition were attenuated in the ad libitum compared with the control groups. However, within ad libitum groups pregnancy outcome parameters were largely unaffected by level or timing of exposure to high protein intakes. The data imply that it is high-energy intakes that are the primary cause of impaired placental development and adverse pregnancy outcome in rapidly growing adolescent sheep.

Effects of Aloe vera on gap junctional intercellular communication and proliferation of human diabetic and nondiabetic skin fibroblasts.

OBJECTIVE: To evaluate the effects of Aloe vera on gap junctional intercellular communication (GJIC) and proliferation of human skin fibroblasts in the presence or absence of basic fibroblast growth factor (FGF-2). DESIGN: In vitro study using human type II diabetic and nondiabetic skin fibroblast cell lines. SETTING AND SUBJECTS: Diabetic (n = 4) and nondiabetic (n = 4) human skin fibroblast cell lines were purchased from Coriell Institute for Medical Research (Camden, NJ). The cells were cultured with or without Aloe vera extract in increasing concentrations (0%, 0.625%, 1.25%, 2.5%, 5%, 10%, and 20%; v/v) in culture medium and with or without FGF-2 (30 ng/mL). MEASUREMENTS: GJIC was evaluated after 48-hour incubation with treatments by laser cytometry. Cells were counted after 72-hour incubation with treatments by using a Coulter counter. RESULTS: The rate of GJIC was greater (p < 0.01) for diabetic than for nondiabetic fibroblasts (3.5 +/- 0.1 versus 3.0 +/- 0.1% per minute during the first 4 minutes after photobleaching). GJIC was increased ( p < 0.05) for diabetic fibroblasts in the presence of 2.5% and 5% of Aloe vera extract (4.2 +/- 0.1 and 4.0 +/- 0.2 versus 3.5 +/- 0.1% per minute for control, respectively). FGF-2 stimulated (p < 0.01) GJIC for diabetic (4.0 +/- 0.1 versus 3.5 +/- 0.1% per minute for control) and nondiabetic (3.5 +/- 0.1 versus 3.0 +/- 0.1% per minute for control) fibroblasts. Aloe vera extract did not affect GJIC of nondiabetic fibroblast cultured without FGF-2. However, Aloe vera extract decreased (p < 0.05) FGF-2 stimulatory effects on GJIC of diabetic and nondiabetic fibroblasts. Proliferation of diabetic fibroblasts was increased (p < 0.05) by 1.25% and 2.5% Aloe vera extract in medium. Proliferation of nondiabetic fibroblasts was not affected by Aloe vera extract. FGF-2 increased (p < 0.05) proliferation of nondiabetic fibroblasts and FGF-2 did not affect proliferation of diabetic fibroblasts. Aloe vera extract decreased (p < 0.05) FGF-2 stimulatory effects on proliferation of nondiabetic fibroblasts. CONCLUSIONS: These data demonstrate that Aloe vera has the ability to stimulate GJIC and proliferation of human skin fibroblasts in diabetes mellitus. Furthermore, these results indicate that Aloe vera contains a compound(s) that neutralizes, binds with FGF-2 receptor, or otherwise alters signaling pathways for FGF-2. By affecting both GJIC and proliferation of diabetic fibroblasts, Aloe vera may improve wound healing in diabetes mellitus.