D.E. Noakes

Mismatched pre- and postnatal nutrition leads to cardiovascular dysfunction and altered renal function in adulthood

The early life environment has long-term implications for the risk of developing cardiovascular (CV) disease in adulthood. Fetal responses to changes in maternal nutrition may be of immediate benefit to the fetus, but the long-term effects of these adaptations may prove detrimental if nutrition in postnatal life does not match that predicted by the fetus on the basis of its prenatal environment. We tested this predictive adaptive response hypothesis with respect to CV function in sheep. We observed that a mismatch between pre- and postnatal nutrient environments induced an altered CV function in adult male sheep that was not seen when environments were similar. Sheep that received postnatal undernutrition alone had altered growth, CV function, and basal hypothalamo–pituitary–adrenal axis activity in adulthood. Prenatal undernutrition induced greater weight gain by weaning compared with the prenatal control diet, which may provide a reserve in the face of a predicted poor diet in later life. In an adequate postnatal nutrient environment (i.e., relatively mismatched), these offspring exhibited cardiac hypertrophy and altered CV function in adulthood. These data support the concept that adult CV function can be determined by developmental responses to intrauterine nutrition made in expectation of the postnatal nutritional environment, and that if these predictions are not met, the adult may be maladapted and at greater risk of CV disease. Our findings have substantial implications for devising strategies to reduce the impact of a mismatch in nutrition levels in humans undergoing rapid socio-economic transitions in both developing and developed societies.

Acute phase protein responses to uterine bacterial contamination in cattle after calving.

Repeated ultrasonographic examinations and collections of blood samples and uterine lumenal swabs between seven and 28 days after calving were used to examine the relative effects of bacterial contamination and involution of the uterus on the concentrations of acute phase proteins in the blood of 26 dairy cows. The severity of bacterial contamination, as determined by the total bacterial growth score, was a significant variable for the concentrations of the acute phase proteins α1-acid glycoprotein (P<0.0001), haptoglobin (P<0.05) and ceruloplasmin (P<0.0001). In addition, the concentrations of α1-acid glycoprotein and ceruloplasmin were increased in the cows from which Escherichia coli (P<0.0001) and Arcanobacterium pyogenes (P<0.05), respectively, were isolated from the uterine lumen. Uterine involution, as determined by the decreasing diameter of the previously gravid uterine horn, was associated with a decrease in the concentrations of α1-acid glycoprotein (P<0.005), haptoglobin (P<0.05) and ceruloplasmin (P<0.01). However, the response of the acute phase proteins to bacterial contamination was independent of the day on which the samples were collected, indicating that their concentrations were increased by bacterial contamination in addition to the changes associated with uterine involution.

UTERINE BACTERIAL FLORA AND UTERINE LESIONS IN BITCHES WITH CYSTIC ENDOMETRIAL HYPERPLASIA (PYOMETRA)

Thirty-eight complete genital tracts were obtained fresh from local veterinary practices after bitches had been ovario-hysterectomised for the treatment of cystic endometrial hyperplasia (pyometra). The aerobic and anaerobic culture of swabs obtained aseptically from the uterine lumen demonstrated the presence of Escherichia coli in 28 of the 29 cases from which bacteria were isolated and, from 25, a pure culture was obtained. Sixteen different serotypes were identified from 24 genital tracts; 032K+ CNF+ was isolated from four of 16 cases and 04K-; from three of 16 cases. Cytotoxin necrotising factors (CNFS) were identified in seven of the 16 serotypes; they included 02, 04, 06, 022, 025, 045 and 075, which except for 06 and 045 were haemolytic. Strains 04K- and 07K+ completely destroyed the integrity of the endometrial epithelium whereas 088K+ and 088K- had the least effect. The scores for the reduction in the integrity of the endometrial epithelium and the degree of infiltration of inflammatory cells were greater in the presence of CNF 1.

Effect of nutritional restriction in early pregnancy on isolated femoral artery function in mid-gestation fetal sheep

Unbalanced maternal nutrition affects fetal endocrine and cardiovascular systems, sometimes accompanied by changes in growth, although this is usually in late gestation. We determined the effect of moderate restriction for the first half of gestation of maternal dietary protein, or of total calorific intake on isolated resistance artery function of mid‐gestation fetal sheep. Welsh Mountain ewes were nutritionally restricted by 30 % of the recommended nutrient intake (globally restricted) or 30 % of the recommended protein intake (protein‐restricted), compared to control ewes fed 100 % of recommended nutrient intake, for ~12 days prior to conception and for the subsequent 70 days of gestation. At mid‐gestation, fetal and placental weights were similar in all dietary groups. In isolated femoral arteries, the response curve to noradrenaline was reduced in protein‐restricted group fetuses (P < 0.05). Maximal relaxation (P < 0.01) and sensitivity (P < 0.05) to acetylcholine were markedly reduced in protein‐restricted group fetuses, and to a smaller extent in globally restricted group fetuses (response curve, P < 0.05). The dilator response (P < 0.05) and sensitivity (P < 0.05) to the α2 agonist UK14304 was lower in protein‐, but not in globally restricted group fetuses. The response (P < 0.05) and sensitivity (P < 0.05) to the nitric oxide donor sodium nitroprusside were reduced in protein‐restricted group fetuses compared to controls. Our data show that dietary imbalance, in particular restricted protein, of the ewe can produce blunting of endothelial‐dependent and ‐independent relaxation in systemic arteries from the mid‐gestation fetus. These changes may precede perturbed late‐gestation fetal and postnatal cardiovascular control.

Ultrasound-guided percutaneous delivery of adenoviral vectors encoding the beta-galactosidase and human factor IX genes to early gestation fetal sheep in utero.

In utero gene therapy may provide treatment of genetic diseases before significant organ damage, allow permanent genetic correction by reaching stem cell populations, and provide immune tolerance against the therapeutic transgenes and vectors. We have used percutaneous ultrasound-guided injection as a minimally invasive fetal procedure. First-generation adenoviruses encoding the nuclear localizing beta-galactosidase reporter gene or the human factor IX (hFIX) gene, or colloidal carbon were delivered via the umbilical vein (UV, n = 4), heart (intracardiac [IC], n = 2), liver parenchyma (intrahepatic [HE], n = 11), peritoneal cavity (intraperitoneal [IP], n = 14), skeletal musculature ([intramuscular [IM], n = 11), or the amniotic cavity (intraamniotic [IA], n = 14) to early-gestation fetal sheep (0.3 gestation = day 33-61). Postmortem analysis was performed at 2, 9, or 28 days after injection. Although fetal survival was between 77% and 91% for IP, HE, IA, and IM routes, no fetuses survived UV or IC procedures. The hFIX levels reaching 1900 and 401 ng/ml (IP), 30 ng/ml (HE), 66.5 and 39 ng/ml (IA), and 83 and 65.5 ng/ml (IM), respectively, were determined 2 days after injection and decreased at birth to 16.5 ng/ml (IP), 7 ng/ml (HE), 4.5 ng/ml (IA), and 4 and 0 ng/ml (IM). Polymerase chain reaction (PCR) and immunohistochemistry showed broadest hFIX transgene spread and highest localised beta-galactosidase expression, respectively, after IP administration. Antibodies were observed against vector but not against hFIX.

Acute phase protein response of ewes and the release of PGFM in relation to uterine involution and the presence of intrauterine bacteria

The rate of uterine involution postpartum was monitored in 13 suckling mule ewes by using radio-opaque markers and radiography, and each ewe was also monitored for intrauterine bacterial contamination during the first week, using a sterile guarded swab. Peripheral plasma or serum concentrations of haptoglobin, seromucoid, ceruloplasmin and 15-keto-13,14-dihydro-prostaglandin F2α (PGFM) were measured up to six weeks postpartum. The maximum reduction in the length of the uterine tody and in the diameters of the homs occurred by 28 days postpartum, except in one ewe in which the size of the uterus continued to decrease for 42 days. Four ewes were positive for intrauterine bacterial contamination; Escheichia coli, clostridial species, Staphylococcus aureus, Streptococcus uberis and Enterococcus species were isolated in pure or mixed culture. The presence of intrauterine bacteria did not affect the time for the completion of uterine involution. No bacteria were isolated from the ewe in which involution was delayed, but it had a different acute phase protein response and was therefore excluded from further analyses. In the remaining 12 ewes the mean postpartum haptoglobin response increased, with peak concentrations occurring on day 1, and decreased slowly as uterine involution progressed, but the four contaminated ewes had a significantly greater response. There was no difference between the prepartum and postpartum concentrations of seromucoid in the eight sterile ewes, but significant increases were observed in the contaminated group; the concentrations of ceruloplasmin did not vary in either group. The concentrations of PGFM were higher during the early postpartum period in the ewes with contaminated uteri.

Peripheral and intrauterine neutrophil function in the cow: the influence of endogenous and exogenous sex steroid hormones.

It has been accepted for many years that the susceptibility of the genital tract to infection is reduced during the follicular phase compared with the luteal phase of the estrous cycle. Since the role of intrauterine neutrophils is paramount in the elimination of bacteria, it can be hypothesized that these differences in resistance to infection could be mediated by differences in uterine-derived neutrophil function. In order to test this hypothesis two groups of cows were used in this study. Group 1 cows (n=5) were studied at estrus, diestrus, after ovariectomy, after exogenous estradiol and after progesterone treatment, at which time they underwent intrauterine infusion with 1% oyster glycogen (OG) and a bacterial-free filtrate (BFF) of Actinomyces genes (BFF), the latter having been recovered from a clinical case of endometritis; neutrophils were harvested by flushing from the lumen 15 to 18 h later. A peripheral blood sample was collected at the time of flushing for the assay of estradiol and progesterone for a WBC and differential count and for the harvesting of neutrophils using a Percoll single-stage discontinuous gradient. After the recovery of the cells they were re-suspended in HBSS. Group 2 (n=4) were infused with BFF during during all reproductive states as Group 1, but with OG only after ovariectomy and after treatment with progesterone and estradiol. Neutrophil chemotaxis was assessed by measuring their migration using a modified Boyden chamber and Zymogen-activated serum as a chemoattractant. Phagocytic activity was measured by determining the number of Candida albicans ingested by each neutrophil after incubation. The percentage of kill was determined using a radiometric assay in which C. albicans was labeled with L-(5-3H) Proline. Peripheral WBC concentration was not influenced by the reproductive state of the cow; however, the mean neutrophil concentration was significantly different between the reproductive states (P<0.001) and between individual cows (P<0.001). In Group 1, there was little difference in the function of the peripheral and uterine neutrophils, and while there were differences in all 3 aspects of neutrophil function from both sources between reproductive states and individual cows, of which some were statistically significant, there was no consistent pattern. In Group 2, neutrophils recovered after the infusion of BFF had poorer function compared with those recovered after the infusion of OG. There was no consistent influence of the reproductive state or individual animal. The hypothesis that the influence of the reproductive state of the cow on the resistance of the uterus to infection is mediated by the inherent differences in either peripheral or intrauterine neutrophil function was not supported by this study.

The effect of maternal undernutrition in early gestation on gestation length and fetal and postnatal growth in sheep

In utero undernutrition in humans may result in cardiovascular (CV), metabolic, and growth adaptations. In sheep, maternal nutrient restriction during pregnancy, without effects on fetal or birth weight, results in altered CV control in the offspring. Adjustment of gestation length after undernutrition could be a strategy to enhance postnatal health/survival. The aim of this study was to determine in sheep the effect of a 50% reduction in maternal nutrient intake [undernutrition group (U) versus 100%, control group (C)] during 1–31 d of gestation (dGA) on gestation length and offspring size. By 28 dGA, U ewes had gained less weight than C, and twin-bearing ewes had gained less weight than singleton-bearing ewes regardless of group (p < 0.05). In different-sex twin pairs, maternal undernutrition resulted in longer gestation compared with C (146.5 ± 0.6 versus 144.6 ± 0.6 d, p < 0.05). Increased weight gain by weaning (20.8 ± 0.8 versus 17.9 ± 0.8 kg, p < 0.05) was observed in U male twins. These findings suggest that the strategy (i.e. growth rate or length of time in utero) adopted by the fetus to enhance immediate survival depends on offspring number and sex. This is likely to reflect the degree of constraint imposed on the fetus.

Sex‐ and age‐specific effects of nutrition in early gestation and early postnatal life on hypothalamo‐pituitary‐adrenal axis and sympathoadrenal function in adult sheep

The early‐life environment affects risk of later metabolic disease, including glucose intolerance, insulin resistance and obesity. Changes in hypothalamo‐pituitary‐adrenal (HPA) axis and sympathoadrenal function may underlie these disorders. To determine consequences of undernutrition in early gestation and/or immediately following weaning on HPA axis and sympathoadrenal function, 2‐ to 3‐year‐old Welsh Mountain ewes received 100% (C, n= 39) or 50% nutritional requirements (U, n= 41) from 1–31 days gestation, and 100% thereafter. From weaning (12 weeks) to 25 weeks of age, male and female offspring were then either fed ad libitum (CC, n= 22; UC, n= 19) or were undernourished (CU, n= 17; UU, n= 22) such that body weight was reduced to 85% of their individual target, based on a growth trajectory calculated from weights taken between birth and 12 weeks. From 25 weeks, ad libitum feeding was restored for all offspring. At 1.5 and 2.5 years, adrenocorticotropic hormone (ACTH) and cortisol concentrations were measured at baseline and in response to corticotropin‐releasing factor (CRF) (0.5 μg kg−1) plus arginine vasopressin (AVP) (0.1 μg kg−1). At 2.5 years, HPA axis and sympathoadrenal (catecholamine) responses to a transport and isolation stress test were also measured. In females, post‐weaning undernutrition reduced pituitary output (ACTH) but increased adrenocortical responsiveness (cortisol:ACTH area under curve) during CRF/AVP challenge at 1.5 years and increased adrenomedullary output (adrenaline) to stress at 2.5 years. In males, cortisol responses to stress at 2.5 years were reduced in those with slower growth rates from 12 to 25 weeks. Early gestation undernutrition was associated with increased adrenocortical output in 2.5‐year‐old females only. Pituitary and adrenal responses were also related to adult body composition. Thus, poor growth in the post‐weaning period induced by nutrient restriction has sex‐ and age‐specific effects on HPA and sympathoadrenal function. With altered glucose tolerance previously reported in this model, this may have long‐term detrimental effects on metabolic homeostasis and cardiovascular function.

Effects of pre- and periconceptional undernutrition on arterial function in adult female sheep are vascular bed dependent

The nutritional environment during development and even prior to conception may contribute to cardiovascular risk. In mature adult female sheep, we investigated the effect of preconceptional and periconceptional maternal nutritional restriction on the vascular reactivity of arteries from four vascular beds supplying the heart, thorax, kidney and hindlimb. Welsh Mountain ewes received 100% of nutrient requirements throughout gestation (control group, C, n= 18), or 50% of nutrient requirements for 30 days prior to conception (preconceptional group, PRE, n= 20) or for 15 days either side of conception (periconceptional group, PERI, n= 31) and 100% thereafter. In 3.5‐year‐old female offspring, the left anterior descending coronary (LAD), left internal thoracic (LITA), right renal and second and third order femoral arteries were dissected and their reactivity was assessed by organ bath or wire myography. Vasoconstrictor responses were greater in both LAD and LITA from PERI offspring compared with C (P < 0.01), while vasoconstriction was unaffected by maternal diet in arteries from the renal and femoral circulations (P= n.s.). Endothelium‐dependent and ‐independent vasodilatation was attenuated in third order femoral arteries of PRE and PERI groups compared with C (P < 0.05). Endothelium‐independent vasodilatation was attenuated in both the LAD and renal arteries in the PERI group compared with C (P < 0.05). These data show that moderate maternal undernutrition either prior to or around conception affects vascular function of adult offspring. The effect depends on the timing of the insult, but also on the vascular bed studied and vessel hierarchy in the vascular tree.