Hepatic steroid inactivating enzymes, hepatic portal blood flow and corpus luteum blood perfusion in cattle.

Production from the corpus luteum (CL) and/or hepatic steroid inactivation impacts peripheral concentrations of P4, which can alter reproductive performance. Our primary objective was to examine hepatic steroid inactivating enzymes, portal blood flow, and luteal blood perfusion at 10 days post-insemination in pregnant versus non-pregnant beef and dairy cows. Twenty early lactation Holstein cows and 20 lactating commercial beef cows were utilized for this study. At day 10 post-insemination, hepatic portal blood flow and CL blood perfusion were measured via Doppler ultrasonography. Liver biopsies were collected and frozen for later determination of cytochrome P450 1A (CYP1A), 2C (CYP2C), 3A (CYP3A), uridine diphosphate-glucuronosyltransferase (UGT) and aldo-keto reductase 1C (AKR1C) activities. Pregnancy was determined at day 30 post-insemination and treatment groups were retrospectively assigned as pregnant or non-pregnant. Data were analyzed using the mixed procedure of SAS. Steroid metabolizing enzyme activity was not different (p > .10) between pregnant versus non-pregnant beef or dairy cows. Hepatic portal blood flow tended (p < .10) to be increased in pregnant versus non-pregnant dairy cows. Luteal blood perfusion was increased (p < .05) in pregnant versus non-pregnant dairy cows. Pregnant dairy cows appear to have an increased rate of hepatic clearance of P4 in combination with increased synthesis from the CL. This could account for the lack of difference in peripheral P4 concentrations between pregnant and non-pregnant dairy cows. This study highlights the relevance of further investigation into steroid secretion and inactivation and their impact on the maintenance of pregnancy in cattle.

Effects of body condition score (BCS) on steroid- and eicosanoid-metabolizing enzyme activity in various mare tissues during winter anoestrus.

The objective of this study was to determine the activity of steroid- and eicosanoid-metabolizing enzymes in horses with varying BCSs. The BCSs of twenty non-pregnant, anoestrous mares were determined prior to euthanasia, and tissue samples were collected from the liver, kidney, adrenal gland, ovary and endometrium. Cytochrome P450 1A (CYP1A), 2C (CYP2C), 3A (CYP3A) and uridine 5'-diphospho-glucuronosyltransferase (UGT) activities were determined using luminogenic substrates. The MIXED procedure of SAS was used to test the effect of BCS on enzyme activity and differences between tissues. Activity of CYP1A in adrenals was increased (p ≤ .05) in BCS 5 versus BCSs 4 and 6. Activity of CYP1A in the liver was increased (p = .05) in BCS 4 versus BCSs 5 and 6. Activity of CYP1A was 100-fold greater (p < .0001) in the liver than in the adrenal, ovary and kidney. Activity of CYP2C was 100-fold greater (p < .0001) in the liver than in the adrenal, ovary and endometrium. Activity of CYP3A was only detectable in the liver. Activity of UGT in the kidney was decreased (p = .02) in BCS 4 versus BCSs 5 and 6. Activity of UGT was threefold greater (p < .0001) in the liver than in the kidney, whereas activity of UGT was ninefold greater (p < .0001) in the kidney than in the ovary and endometrium. In general, BCS did not alter the activity of steroid- and eicosanoid-metabolizing enzymes in horses. However, tissue differences in these enzymes indicated abundant hepatic metabolism in horses, which is similar to other livestock species.

Effects of realimentation after nutrient restriction during mid- to late gestation on pancreatic digestive enzymes, serum insulin and glucose levels, and insulin-containing cell cluster morphology.

This study examined effects of stage of gestation and nutrient restriction with subsequent realimentation on maternal and foetal bovine pancreatic function. Dietary treatments were assigned on day 30 of pregnancy and included: control (CON; 100% requirements; n = 18) and restricted (R; 60% requirements; n = 30). On day 85, cows were slaughtered (CON, n = 6; R, n = 6), remained on control (CC; n = 12) and restricted (RR; n = 12), or realimented to control (RC; n = 11). On day 140, cows were slaughtered (CC, n = 6; RR, n = 6; RC, n = 5), remained on control (CCC, n = 6; RCC, n = 5) or realimented to control (RRC, n = 6). On day 254, the remaining cows were slaughtered and serum samples were collected from the maternal jugular vein and umbilical cord to determine insulin and glucose concentrations. Pancreases from cows and foetuses were removed, weighed, and subsampled for enzyme and histological analysis. As gestation progressed, maternal pancreatic α-amylase activity decreased and serum insulin concentrations increased (p ≤ 0.03). Foetal pancreatic trypsin activity increased (p < 0.001) with advancing gestation. Foetal pancreases subjected to realimentation (CCC vs. RCC and RRC) had increased protein and α-amylase activity at day 254 (p ≤ 0.02), while trypsin (U/g protein; p = 0.02) demonstrated the opposite effect. No treatment effects were observed for maternal or foetal pancreatic insulin-containing cell clusters. Foetal serum insulin and glucose levels were reduced with advancing gestation (p ≤ 0.03). The largest maternal insulin-containing cell cluster was not influenced by advancing gestation, while foetal clusters grew throughout (p = 0.01). These effects indicate that maternal digestive enzymes are influenced by nutrient restriction and there is a potential for programming of increased foetal digestive enzyme production resulting from previous maternal nutrient restriction.

Effects of Interferon-Tau and Steroids on Cytochrome P450 Activity in Bovine Endometrial Epithelial Cells.

The objective of the current study was to examine cyclooxygenase (COX), cytochrome P450 1A (CYP1A) and 2C (CYP2C) activity in bovine endometrial cell cultures following exposure to oxytocin (OT), interferon-τ (IFN), estradiol (E2) and/or progesterone (P4). Bovine endometrial epithelial cells were treated with OT, IFN, a combination of OT+IFN or control (CON) media for 24 h. For the second experiment, cells were treated with E2, P4, a combination of E2 + P4 or CON media for 24 h. Treatments were performed in triplicate, and the experiment was repeated four times (n = 12 per treatment). Treatment with OT alone increased (p < 0.01) activity of COX compared with CON; however, OT alone did not alter activity of CYP1A (p = 0.55) or CYP2C (p = 0.46) compared with CON. Activity of CYP1A and CYP2C was decreased in cells exposed to IFN (p < 0.01) or OT+IFN (p < 0.01) compared with CON. Treatment with E2 alone did not alter activity of CYP1A (p = 0.64) or CYP2C (p = 0.06) compared with CON. Activity of CYP1A and CYP2C was decreased (p < 0.01) in P4 vs CON. In summary, IFN exposure, irrespective of OT treatment, decreased the activity of CYP1A and CYP2C. Activity of CYP1A was decreased following P4 treatment alone, while that of CYP2C was decreased following both P4 and E2 + P4 treatment. The mixed function monooxygenase enzymes, CYP1A and CYP2C, have been implicated in synthesizing embryotoxic compounds; therefore, downregulation in the endometrium may be necessary during maternal recognition of pregnancy.

Effects of Supplementing Holstein Heifers with Dietary Melatonin during Late Gestation on Growth and Cardiovascular Measurements of their Offspring.

The objective was to examine the effects of supplementing dams with dietary melatonin during late gestation on offspring growth and cardiovascular measurements. On day 190 of gestation, heifers (n = 20) were blocked by body weight and randomly assigned to one of two dietary treatments consisting of 20 mg of dietary melatonin per day [melatonin (MEL)] or no melatonin supplementation [control (CON)]. Dietary treatments were terminated on day 262 of gestation. At birth, calves were separated from their dams with no further treatments. Calf (n = 18) blood pressure, cortisol, nitrites and total antioxidant capacity were collected on weeks 0, 1, 2, 3 and 4 of age. Calf hepatic portal blood flow and concentrations of insulin-like growth factor 1 were determined on weeks 0 and 4 of age. Calf body weight, abdominal girth, hip height and wither height increased (p < 0.05) with age. An age by treatment interaction (p < 0.01) was observed for calf body weight, which was increased at weeks 8 and 9 of age in calves born to MEL heifers compared to calves born to CON heifers. Pulse pressure, mean arterial pressure, absolute hepatic portal blood flow and blood flow relative to calf body weight were not different (p > 0.05) between treatments. A main effect of calf age (p < 0.05) was observed for concentrations of insulin-like growth factor 1, which was decreased at week 4 compared to week 0. An age by treatment interaction (p < 0.05) was observed for cortisol, which was decreased at week 2 in calves from MEL-treated dams compared to calves from CON-treated dams. Early post-natal growth was altered in offspring born to dams supplemented with dietary melatonin. This could lead to further foetal programming implications in conjunction with post-natal development.

Effect of 2 corpora lutea on blood perfusion, peripheral progesterone, and hepatic steroid-inactivating enzymes in dairy cattle.

The luteal structure that develops postovulation is critical to the facilitation and maintenance of pregnancy in dairy cattle. The objectives of this experiment were to determine if the induction of an accessory corpus luteum (CL), via human chorionic gonadotropin, altered blood perfusion of CL, peripheral concentrations of progesterone, or hepatic steroid-inactivating enzymes. Twenty-eight late-lactation Holstein cows were synchronized using the Ovsynch protocol and randomly assigned to 1 of 2 treatment groups. Cows received either an injection of human chorionic gonadotropin (1,000IU, i.m.) to induce an accessory CL (cows had exactly 2CL in 1 ovary) or no treatment (cows had exactly 1CL). Corpora lutea were examined daily from d 10 to 18 (d 0 was induced ovulation) via Doppler ultrasonography and a blood sample was collected. Volume of the CL was recorded, as well as images and videos of each CL, which were analyzed for blood perfusion. On d 13, a liver biopsy was performed to analyze hepatic steroid-inactivating enzymes. Cows with 1 or 2CL had similar peripheral concentrations of progesterone. Cows with 2CL had similar luteal volumes to cows with 1CL but cows with 2CL had greater total luteal blood perfusion. Hepatic enzyme [cytochrome P450 (CYP) 1A, 3A, and 2C, aldo-keto reductase 1C, and uridine diphosphate glucuronosyltransferase] activities did not differ between cows with 1 and 2CL. Overall, the observed increase in total luteal blood perfusion in cows with 2CL did not correspond to differences in peripheral concentrations of progesterone or clearance of progesterone measured by the hepatic enzyme activity. This could indicate that induction of an accessory CL would not affect concentrations of progesterone necessary to maintain pregnancy.

Effects of nutrient restriction and melatonin supplementation on maternal and foetal hepatic and small intestinal energy utilization.

To determine how nutrient restriction and melatonin supplementation influence ewe and foetal hepatic and small intestinal energy use, 32 primiparous ewes on d 50 of gestation were fed 60% (RES) or 100% (ADQ) of NRC recommendations with 0 (CON) or 5 mg/d (MEL) of dietary melatonin. On d 130 of gestation, small intestine and liver were weighed and collected. Data were analysed as a completely randomized design with a 2 × 2 factorial arrangement of treatments. Liver weight (g/kg EBW) decreased (p = 0.02) in RES ewes. Jejunum weight (g/kg BW) increased (interaction p = 0.04) in ADQ-MEL ewes compared with all other treatments. Total in vitro O2 consumption (mol/min/tissue) and total citrate synthase activity (mol/min/tissue and mol/min/kg EBW) in liver decreased (p ≤ 0.03) in RES ewes. Oxygen consumption (mol/min/kg EBW) increased (interaction p = 0.02) in jejunum of ADQ-CON versus RES-MEL and ADQ-CON. Citrate synthase activity (mol/min/kg of EBW) increased (interaction p = 0.03) in jejunum of ADQ-MEL compared with RES-MEL and ADQ-CON. Foetal liver weight (g/kg BW) decreased (p = 0.02) in RES versus ADQ. Foetal small intestine weight (g/kg BW) decreased (interaction p = 0.05) in RES-MEL versus ADQ-MEL. Total O2 consumption (mol/min/tissue) and total citrate synthase activity (mol/min/kg of BW) in foetal liver decreased (p ≤ 0.05) in RES versus ADQ. Foetal small intestinal O2 consumption (mol/min/kg of BW) was greater (interaction p = 0.03) in RES-CON and ADQ-MEL than RES-MEL and ADQ-CON. Maternal nutrient restriction had a greater effect than melatonin supplementation on liver and jejunum mass and energy utilization in dams and foetuses. Because intestinal mass and energy utilization were more responsive to melatonin supplementation in ewes fed adequate nutrition compared with restricted ewes, melatonin may have limited use as a therapeutic supplement to help overcome potential negative effects of nutrient restriction.

Characterization of placentome vascular perfusion in relation to pregnancy associated glycoproteins throughout gestation in pregnant beef heifers.

During pregnancy, blood flow to the uterus changes to support fetal demand. Placentomes serve as vascular attachment sites on the placenta for exchange of gases, nutrients, and metabolic products. Non-invasive methods of ultrasonography and biomarkers have been described to assess placental health and fetal viability. Pregnancy associated glycoproteins (PAGs) are produced by the ruminant placenta and are detected in maternal circulation. In cattle, changes in circulating PAG concentrations are associated with embryonic and fetal outcomes. The objective of this study was to determine the association between placentome blood perfusion and circulating PAG concentrations as they relate to the health of the developing fetus. We hypothesized that placentome perfusion and PAG concentration will be positively correlated and associated with neonatal outcome. A prospective, observational study was designed using 26 pregnant, nulliparous, Angus heifers in which PAG concentration and placentome blood perfusion were assessed throughout gestation, with assessment of calving characteristics following parturition. Placentome blood perfusion was visualized at 30-day intervals via transrectal Doppler ultrasonography with power flow function. Ultrasound images were analyzed using ImageJ software to determine the percent area of perfusion and integrated pixel densities. Venous blood was collected and PAG concentrations were determined via serum PAG enzyme-linked immunoassay. Mean placentome blood perfusion increased as gestation advanced. PAG concentrations demonstrated the expected temporal trend, increasing with gestation length, and were positively linearly correlated with placentome perfusion (P < 0.0001). The relationship identified between circulating PAG concentration and placentome blood perfusion suggests the use of transrectal power flow Doppler ultrasonography as a noninvasive technique to determine placental blood flow morphometrics to assess conceptus wellbeing throughout pregnancy.

Effects of nutrient restriction and subsequent realimentation in pregnant beef cows: Maternal endocrine profile, umbilical hemodynamics, and mammary gland development and hemodynamics.

Our hypothesis was that maternal nutrient restriction would negatively impact the endocrine and metabolic status of the pregnant cow, therefore influencing the mammary gland in preparation for lactation. We further hypothesized that earlier timing of realimentation could prevent negative impacts of nutrient restriction. The objectives were to investigate the influence of nutrient restriction and realimentation during early to late gestation on endocrine profile, umbilical hemodynamics, and mammary gland development and hemodynamics in pregnant beef cows. In Experiment 1, on d 30 of pregnancy cows (initial BW = 667.5 ± 13.4 kg, BCS = 6.2 ± 0.1) were randomly assigned to one of 3 treatments: 1) 100% NRC requirements from d 30 to 254 of gestation (CCC; n = 6); 2) 60% NRC from d 30 to 85, thereafter being re-alimented to 100% NRC to d 254 (RCC; n = 5); 3) or receive 60% NRC from d 30 to 140, thereafter being re-alimented to 100% NRC to d 254 (RRC; n = 6). Cows were returned to a common outdoor facility for calving thereafter and were fed ad libitum. In Experiment 2, on d 30 of pregnancy, cows (initial BW = 620.5 ± 11.3 kg, BCS = 5.1 ± 0.1) were randomly assigned to dietary treatments including: control (CON; 100% NRC; n = 18) and nutrient restriction (RES; 60% NRC; n = 30). On d 85 of pregnancy, cows were either slaughtered (CON, n = 6 and RES, n = 6), remained on control (CC; n = 12) and restricted (RR; n = 12) treatments, or were realimented to control (RC; n = 11). On d 140 of pregnancy, cows were either slaughtered (CC, n = 6; RR, n = 6; RC, n = 5), remained on control (CCC, n = 6; RCC, n = 5), or were realimented to control (RRC, n = 6). On d 254 of pregnancy, all remaining cows were slaughtered (CCC, n = 6; RCC, n = 5; RRC, n = 6). Mammary hemodynamics and endocrine profile were measured. Serum urea nitrogen, NEFA, as well as fetal parameters were measured in Experiment 1; whereas in Experiment 2, mammary gland development was recorded. In Experiment 1, RRC cows had lower dry matter intake (P = 0.001) and consequently lower BW change (P = 0.06). However, maternal nutrition did not alter mammary hemodynamics, hormonal patterns, and fetal characteristics (P > 0.11). In Experiment 2, CCC cows had increased (P = 0.02) mammary gland blood flow ipsilateral to the gravid horn as well as greater (P = 0.02) mammary gland fat on d 254. Nevertheless, plane of nutrition did not alter hormonal concentrations nor mammary gland characteristics (P > 0.15). These data indicate that nutrient restriction did not alter mammary hemodynamics nor endocrine profile throughout gestation.

Effects of nutrient restriction and melatonin supplementation from mid-to-late gestation on maternal and fetal small intestinal carbohydrase activities in sheep.

The objective of this experiment was to evaluate the effects of nutrient restriction and melatonin supplementation during mid-to-late gestation on maternal and fetal small intestinal carbohydrase activities in sheep. Ewes were randomly assigned to one of 4 dietary treatments arranged in a 2 × 2 factorial design. Ewes were fed to provide 100% (adequate; ADQ) or 60% (restricted; RES) of nutrient recommendations, and diets were supplemented with either no melatonin (control; CON) or 5 mg melatonin/d (melatonin; MEL). This resulted in 4 treatment groups: CON-ADQ (n = 7), CON-RES (n = 8), MEL-ADQ (n = 8), MEL-RES (n = 8). Treatments began on day 50 of gestation, and ewes were euthanized on day 130 for tissue collection. The maternal and fetal small intestine were collected and assayed for small intestinal carbohydrase activities. Data were analyzed using the GLM procedure of SAS with fetal sex, melatonin, nutrition, and the melatonin by nutrition interaction included in the model statement. There were no melatonin by nutrition interactions for maternal or fetal small intestinal protein concentration or carbohydrase activities (P ≥ 0.11). Dietary melatonin supplementation decreased (P = 0.03) maternal small intestinal protein concentration by 22.7% and increased (P = 0.03) maternal small intestinal glucoamylase, isomaltase, and maltase activity per gram protein by 45.5%, 41.3%, and 40.6%, respectively. Nutrient restriction from mid-to-late gestation did not influence (P ≥ 0.46) maternal small intestinal protein concentration, or maltase, isomaltase, and lactase activity. Maternal glucoamylase activity per gram intestine increased (P = 0.05) with nutrient restriction by 49.1%. Melatonin supplementation and maternal nutrient restriction did not influence (P ≥ 0.15) fetal small intestinal protein concentration, or glucoamylase, isomaltase, and lactase activity. Maternal nutrient restriction from mid-to-late gestation decreased (P = 0.05) fetal maltase activity per gram intestine by 20.5% but did not influence fetal maltase activity per gram protein. These data indicate that some maternal and fetal carbohydrases are influenced by nutrient restriction and melatonin supplementation in sheep. More information is needed to understand how nutritional and hormonal factors regulate digestive enzyme activity in ruminants to design improved maternal nutrition programs to optimize fetal growth and development while maintaining maternal productivity.

Effect of cytochrome P450 and aldo-keto reductase inhibitors on progesterone inactivation in primary bovine hepatic cell cultures.

Progesterone is required for maintenance of pregnancy, and peripheral concentrations of progesterone are affected by both production and inactivation. Hepatic cytochrome P450 (EC 1.14.14.1) and aldo-keto reductase (EC 1.1.1.145-151) enzymes play a pivotal role in the first step of steroid inactivation, which involves the addition of hydroxyl groups to various sites of the cyclopentanoperhydrophenanthrene nucleus. The current objective was to discern the proportional involvement of hepatic progesterone inactivating enzymes on progesterone decay using specific enzyme inhibitors. Ticlopidine, diltiazem, curcumin, dicumarol, and naproxen were used because of their selective inhibition of cytochrome P450s, aldo-keto reductases, and glucuronosyltransferases. Liver biopsies were collected from 6 lactating Holstein dairy cows, and cells were dissociated using a nonperfusion technique. Confluent wells were preincubated for 4 h with enzyme inhibitor and then challenged with progesterone for 1 h. Cell viability was unaffected by inhibitor treatment and averaged 84±1%. In control wells, 50% of the progesterone had been inactivated after a 1-h challenge with 5 ng/mL of progesterone. Preincubation with curcumin, ticlopidine, or naproxen caused the greatest reduction in progesterone inactivation compared with controls and averaged 77, 39, or 37%, respectively. Hydroxylation of 4-nitrophenol to 4-nitrocatechol in intact cells was inhibited by approximately 65% after treatment with curcumin or ticlopidine. Glucuronidation of phenol red or 4-nitrocatechol in intact cells was inhibited by treatment with curcumin, dicumarol, or naproxen. In cytoplasmic preparations, aldo-keto reductase 1C activity was inhibited by curcumin, dicumarol, or naproxen treatment. Microsomal cytochrome P450 2C activity was inhibited by treatment with curcumin or ticlopidine, whereas cytochrome P450 3A activity was inhibited by treatment with curcumin or diltiazem. The contribution of cytochrome P450 2C and cytochrome P450 3A enzymes to progesterone inactivation in bovine hepatic cell cultures was 40 and 15%, respectively. Depending on the inhibitor used, it would appear that the aldo-keto reductase enzymes contribute approximately 40% to the observed progesterone inactivation, although a portion of this inactivation may be attributed to the loss of glucuronosyltransferase activity. Future work focusing on decreasing the activity of these enzymes in vivo could lead to an increase in the bioavailability of progesterone.

Effect of a high cornstarch diet on hepatic cytochrome P450 2C and 3A activity and progesterone half-life in dairy cows.

In the cow, inadequate concentrations of progesterone during gestation may lead to an abrupt termination of pregnancy. The primary organ involved in progesterone catabolism is the liver, which contains an abundance of cytochrome P450 isozymes (EC 1.14.14.1; mixed-function monooxygenases). The objectives of the current experiment were to determine the effect of feeding 2 isoenergetic and isonitrogenous diets, formulated to cause divergent insulin secretion, on hepatic cytochrome P450 2C (CYP2C) and 3A (CYP3A) activity as well as the resulting biological half-life of progesterone. Twenty-two Holstein cows averaging 80+/-7 d in milk were randomly assigned to either a high cornstarch diet or a high fiber diet in a crossover experimental design consisting of two 14-d periods. Dry matter intake, milk yield, milk lactose yield, and milk lactose percentage were similar between the 2 diets. Milk fat yield and milk fat percentage were higher in cows fed the high fiber diet, whereas milk protein yield tended to be higher and milk protein percentage was higher in cows fed the high cornstarch diet. Energy balance tended to be improved by 57% in cows consuming the high cornstarch diet. Insulin concentrations at the time of liver biopsy (3.16+/-0.04h post-feeding) were increased by 44% in cows consuming the high cornstarch diet compared with cows consuming the high fiber diet. Cytochrome P450 2C activity was decreased by 45%, whereas CYP3A activity tended to be lowered by 34% in cows consuming the high cornstarch diet. Cytochrome P450 2C mRNA expression tended to be decreased by 21% in cows fed the high cornstarch diet, whereas CYP3A mRNA expression was not different between the dietary treatments. The fractional rate constant of progesterone decay was not different between the 2 diets; however, the half-life of progesterone tended to be longer in cows fed the high cornstarch diet compared with cows fed the high fiber diet (85 vs. 64min, respectively). In summary, cows consuming the high cornstarch diet had increased insulin concentrations and lower hepatic CYP2C and CYP3A activity and tended to have a longer progesterone half-life compared with cows consuming the high fiber diet. Feeding diets that stimulate insulin secretion could alter progesterone clearance during lactation, when dairy cows have increased rates of progesterone inactivation because of high energy demands and increased DMI.

Short communication: insulin alters hepatic progesterone catabolic enzymes cytochrome P450 2C and 3A in dairy cows.

High proportions of embryonic and early fetal losses in dairy cattle are associated with low peripheral concentrations of progesterone, which could result from increased catabolism, decreased production, or both. Progesterone catabolism occurs primarily in the liver via the cytochrome P450 2C (CYP2C) and cytochrome P450 3A (CYP3A) subfamilies (EC 1.14.14.1; unspecific monooxygenases). Recent observations from our laboratory have shown that the fractional rate constant of progesterone decay can be dramatically reduced by insulin because of a decrease in hepatic CYP2C and CYP3A activity. Little information exists on the regulation of progesterone catabolic enzymes in dairy cows. We hypothesized that elevated insulin concentrations would down-regulate hepatic CYP2C and CYP3A mRNA; therefore, our objectives were to determine the relative abundance of hepatic CYP2C and CYP3A mRNA in dairy cows in response to elevated concentrations of insulin. In the first experiment, 17 mature Holstein cows were drenched daily with 500 mL of water (n = 10) or propylene glycol (a gluconeogenic substrate; n = 7) from 10 d before their expected calving date until d 25 postpartum. Cows drenched with propylene glycol had a 30% increase in peripheral concentrations of insulin. Liver biopsies were collected on d 25 postpartum to determine the relative abundance of CYP2C and CYP3A mRNA. In the second experiment, 19 mature, lactating Holstein cows were randomly assigned to a hyperinsulinemic-euglycemic clamp (0.3 or 1.0 microg of insulin/kg of BW per h; n = 6 each) or remained as controls (saline infused; n = 7) for 96 h beginning on d 10 postpartum. Insulin infusion resulted in a 2.6- or 8- fold increase in peripheral concentrations of insulin, respectively. On d 14 postpartum, a liver biopsy was collected to determine CYP2C and CYP3A mRNA abundance. In experiment 1, the relative abundance of CYP2C mRNA in cows treated with propylene glycol did not differ from controls; however, the relative abundance of CYP3A mRNA in the propylene glycol group was 63% that of controls. For experiment 2, there was a dose-dependent decrease in the relative abundance of both CYP2C and CYP3A mRNA with increasing dosage of insulin. In conclusion, this study demonstrated that, in the cow, either providing a gluconeogenic feed-stuff or treatment with insulin decreased the abundance of mRNA for enzymes responsible for hepatic progesterone catabolism.

Uteroplacental secretion of progesterone and estradiol-17ß in an ovine model of intrauterine growth restriction.

Using a mid to late gestation model of intrauterine growth restriction, uteroplacental secretion of progesterone and estradiol-17ß were examined. From day 50 to 130 of gestation, 31 ewe lambs were allocated to receive 100% (ADQ) or 60% (RES) of nutrient requirements. At day 130, umbilical and uterine artery blood flows were determined and blood samples were collected from maternal saphenous artery, gravid uterine vein, umbilical vein, and umbilical artery. Uteroplacental secretion of progesterone was increased in RES compared to ADQ fed dams. There was a net secretion and net metabolism of estradiol-17ß in RES, and ADQ fed dams, respectively. In relation to steroid synthesis, cotyledonary abundance of steroidogenic acute regulatory protein was greater in RES compared with ADQ fed dams, while abundance of aromatase was not different between dietary treatments. Caruncular aldo-keto reductase 1C abundance was less in RES compared to ADQ fed dams. The increase in progesterone secretion, therefore, is due in part to an increase in synthesis and a decrease in placental catabolism. Caruncular cytochrome P450 3A, which catalyzes the conversion of estrogens to catechol-estrogens, was in lesser abundance in RES compared to ADQ fed dams. Opposite responses in estradiol-17ß uteroplacental secretion compared with metabolism may be mediated through placental estrogen metabolism via cytochrome P450 enzymes.

Endometrial blood perfusion as assessed using a novel laser Doppler technique in Angus cows.

Previous studies have characterized ovarian steroid synthesis which directly affects uterine environment and blood flow. Clearance of steroids occurs primarily in hepatic tissues, however, it was discovered that there is an abundant activity of the phase II steroid metabolizing enzyme UDP-glucuronosyltransferase (UGT) in uterine biopsies. No minimally invasive techniques for collecting endometrial perfusion, which is affected by steroids and indicative of reproductive health, have been developed for livestock. The objective of the present study was to characterize UGT activity and endometrial blood perfusion during a normal estrous cycle of cattle. It was hypothesized that there would be increased steroid metabolism during the luteal phase of the estrous cycle and in the uterine horn ipsilateral to the corpus luteum (CL). During the first synchronized estrous cycle, progesterone and UGT activity increased on Day 6 compared with 0 and 3, with the first day of estrus being considered Day 0 of the study. Endometrial perfusion was greater ipsilateral to the CL compared with contralateral on Day 12, and was less ipsilateral to the CL compared with contralateral on Day 18. Similar to perfusion results, nitric oxide metabolites (nitrites) were greatest in the endometrium ipsilateral as compared with contralateral to the CL. Moreover, there was a positive correlation (r = 0.28; P = .04) between endometrial perfusion and nitrite concentration. It is concluded that activity of UGT within the endometrium is affected by the contralateral or ipsilateral location of the CL, and collection of endometrial perfusion data using a laser Doppler probe could be a viable measurement technique as indicated by associated nitrite concentrations in the present study.

Effects of maternal nutrient restriction followed by realimentation during early and mid-gestation in beef cows. II. Placental development, umbilical blood flow, and uterine blood flow responses to diet alterations.

The objectives were to examine the effects of maternal nutrient restriction followed by realimentation during early to mid-gestation on placental development and uterine and umbilical hemodynamics in the beef cow. On day 30 of pregnancy, multiparous, non-lactating beef cows (620.5 ±â€¯11.3 kg) were assigned to 1 of 2 dietary treatments: control (C; 100% National Research Council [NRC] recommendations; n = 18) and restricted (R; 60% NRC; n = 30). On day 85, cows were slaughtered (C, n = 6; R, n = 6), remained on control (CC; n = 12) and restricted (RR; n = 12), or were realimented to control (RC; n = 11). On day 140, cows were slaughtered (CC, n = 6; RR, n = 6; RC, n = 5), remained on control (CCC, n = 6; RCC, n = 5), or were realimented to control (RRC, n = 6). On day 254, all remaining cows were slaughtered. Heart rate and umbilical and uterine hemodynamics [blood flow, resistance index (RI), and pulsatility index (PI)] were determined via Doppler ultrasonography. As expected umbilical blood flow increased and fetal heart rate decreased as gestation advanced. Umbilical PI in RRC cows was less (P = 0.01) compared to RCC and CCC. During late gestation, RCC cows had greater (P = 0.02) ipsilateral and total uterine blood flow vs. CCC and RRC. There was an increase in the number and weight of placentomes from R cows (P ≤ 0.02) compared to C cows (i.e. day 85). There were more placentomes (P = 0.03) in RR vs. CC and RC cows, but placentome weight was not affected (P = 0.18) by maternal dietary treatment at day 140. Maternal nutrient restriction during early to mid-gestation increased the weight (by day 85) and number (day 85 and 140) of placentomes, and did not reduce fetal weight compared to control cows. A longer realimentation period may enhance uterine blood flow and individual placentome size during later gestation, which may compensate for reduced nutrients experienced early in gestation.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Alterations in uteroplacental hemodynamics during melatonin supplementation in sheep and cattle.

Compromised placental function can result in fetal growth restriction which is associated with greater risk of neonatal morbidity and mortality. Large increases in transplacental nutrient and waste exchange, which support the exponential increase in fetal growth during the last half of gestation, are dependent primarily on the rapid growth and vascularization of the uteroplacenta. The amplitude of melatonin secretion has been associated with improved oxidative status and altered cardiovascular function in several mammalian species; however, melatonin mediated alterations of uteroplacental capacity in sheep and cattle are lacking. Therefore, our laboratories are examining uteroplacental blood flow and fetal development during maternal melatonin supplementation. Using a mid- to late-gestation ovine model of intrauterine growth restriction, we examined uteroplacental blood flow and fetal growth during supplementation with 5 mg/d of dietary melatonin. Maternal nutrient restriction decreased uterine arterial blood flow, while melatonin supplementation increased umbilical arterial blood flow compared with non-supplemented controls. Although melatonin treatment did not rescue fetal weight in nutrient restricted ewes; we observed disproportionate fetal size and fetal organ development. Elevated fetal concentrations of melatonin may result in altered blood flow distribution during important time points of development. These melatonin specific responses on umbilical arterial hemodynamics and fetal development may be partially mediated through vascular melatonin receptors. Recently, we examined the effects of supplementing Holstein heifers with 20 mg/d of dietary melatonin during the last third of gestation. Uterine arterial blood flow was increased by 25% and total serum antioxidant capacity was increased by 43% in melatonin supplemented heifers vs. non-supplemented controls. In addition, peripheral concentrations of progesterone were decreased in melatonin supplemented heifers vs. non-supplemented controls. Using an in vitro model, melatonin treatment increased the activity of cytochrome P450 2C, a progesterone inactivating enzyme, which was blocked by treatment with the melatonin receptor antagonist, luzindole. Elucidating the consequences of specific hormonal supplements on the continual plasticity of placental function will allow us to determine important endogenous mediators of offspring growth and development.

The effect of follicular wave on fertility characteristics in beef cattle.

The objectives of this experiment were to determine the effects of follicular wave (first or second) on diameter of the dominant follicle, concentrations of progesterone and estradiol and the hepatic enzymes that inactivate them, thickness of the endometrium, and pregnancy rates to AI. Beef heifers ( = 101) and cows ( = 106) were randomly assigned to 1 of 2 treatments: insemination to the first follicular wave (FFW) or insemination to the second follicular wave (SFW). Estrous cycles of females were synchronized to ensure appropriate timing for the treatments. The MIXED procedure of SAS was used for analysis. A similar proportion of females in each treatment responded to presynchronization; however, females in the FFW group ovulated in response to the first injection of GnRH of the CO-Synch protocol more frequently. Only females ( = 94) that properly responded to ovulation synchronization were included in further analyses. Cows in the FFW group tended ( 0.06) to have larger ovulatory follicles 36 h post-PGF of the CO-Synch protocol compared to cows in the SFW group (14.22 ± 0.42 and 11.83 ± 0.49, respectively), whereas heifers were similar between treatment groups. Three d prior to AI, circulating concentrations of progesterone were lesser ( 0.01) in females in the FFW (3.63 ± 0.80 ng/mL) than in the SFW (7.12 ± 0.83 ng/mL), whereas concentrations of estradiol tended ( 0.08) to be greater in those in the FFW (82.72 ± 6.48 pg/mL) than in the SFW (65.55 ± 6.74 pg/mL). Concentrations of cytochrome P450 1A in the liver were lesser ( 0.01) in females in the FFW than those in the SFW (0.68 ± 0.08 vs. 0.96 ± 0.06, respectively). Endometrial thicknesses were similar between treatments but were thicker ( < 0.0001) in cows (9.73 ± 0.24 mm) than heifers (7.22 ± 0.26 mm). When considering all females or only those that were properly presynchronized, pregnancy rates were similar between treatments. However, when evaluating females that ovulated to the assigned follicular wave, there was a treatment by parity interaction ( = 0.04) with heifers in the FFW having a lesser pregnancy rate (25.9%) than heifers in the SFW (72.0%) while cows in both treatment groups were intermediate (45.4% in FFW and 50.0% in SFW). The differences in concentrations of steroids between treatment groups may affect fertility of heifers; however, additional research is necessary.

Dietary melatonin alters uterine artery hemodynamics in pregnant Holstein heifers.

The objective was to examine uterine artery hemodynamics and maternal serum profiles in pregnant heifers supplemented with dietary melatonin (MEL) or no supplementation (CON). In addition, melatonin receptor-mediated responses in steroid metabolism were examined using a bovine endometrial epithelial culture system. Twenty singleton pregnant Holstein heifers were supplemented with 20 mg of melatonin (n = 10) or no melatonin supplementation (control; n = 10) from days 190 to 262 of gestation. Maternal measurements were recorded on days 180 (baseline), 210, 240, and 262 of gestation. Total uterine blood flow was increased by 25% in the MEL-treated heifers compared with the CON. Concentrations of progesterone were decreased in MEL vs CON heifers. Total serum antioxidant capacity was increased by 43% in MEL-treated heifers when compared with CON. Activity of cytochrome P450 1A, 2C, and superoxide dismutase was increased in bovine endometrial epithelial cells treated with melatonin, whereas the melatonin receptor antagonist, luzindole, negated the increase in cytochrome P450 2C activity. Moreover, estradiol or progesterone treatment altered bovine uterine melatonin receptor expression, which could potentiate the melatonin-mediated responses during late gestation. The observed increase in total uterine blood flow during melatonin supplementation could be related to its antioxidant properties. Compromised pregnancies are typically accompanied by increased oxidative stress; therefore, melatonin could serve as a therapeutic supplementation strategy. This could lead to further fetal programming implications in conjunction with offspring growth and development postnatally.

Evaluating blood perfusion of the corpus luteum in beef cows during fescue toxicosis.

The aim of this study was to determine if fescue toxicosis altered blood perfusion in the corpus luteum (CL) and peripheral concentrations of progesterone in cattle. The estrous cycles of 36 nonpregnant Angus or Charolais cows were synchronized in 2 replicates using the CO-Synch+CIDR protocol. Seven days after initiation of the protocol, cows were assigned (d 0) to 1 of 2 dietary treatments: 2.5 kg of 1) Kentucky-31 endophyte-infected (KY31; = 14) or 2) MaxQ novel endophyte (MaxQ; = 12) tall fescue seed. On d 7, ovaries were examined using ultrasonography, and only cows that had 1 CL present remained on the study ( = 26). Images of blood perfusion of CL, blood samples, rectal temperatures, and blood pressure of tails were collected on d 10, 13, 15, and 18. Images of CL blood perfusion were analyzed using ImageJ software for pixel density, and scored visually (0 to 9 with 0 = no perfusion, 9 = complete perfusion) by 2 independent technicians. The MIXED procedure of SAS was used with day as a repeated measure. Least squares means and SEM are reported. Cows receiving KY31 had greater rectal temperatures ( 0.003; 38.76 ± 0.08°C) than those receiving MaxQ (38.44 ± 0.08°C), providing evidence that the cows treated with KY31 were influenced by fescue toxicosis. Pulse pressure and mean arterial pressure were decreased ( < 0.01) in cows receiving KY31 (55.26 ± 2.81 and 80.06 ± 2.72 mmHg, respectively) than MaxQ (66.58 ± 3.03 and 91.38 ± 2.93 mmHg, respectively). Concentrations of progesterone were similar ( = 0.54) between cows receiving KY31 (6.04 ± 0.53 ng/mL) or MaxQ (6.36 ± 0.63 ng/mL). Pixel densities ( = 0.14) and visual perfusion scores were similar ( = 0.11) between cows receiving KY31 (1477.20 ± 655.62 pixels and 2.23 ± 0.34, respectively) or MaxQ (2934.70 ± 718.20 pixels and 3.00 ± 0.36, respectively). Mean CL volume was similar ( 0.95) between treatments. In conclusion, blood perfusion of CL or peripheral concentrations of progesterone were not altered at the onset of fescue toxicosis in this short-term study, indicating that a decrease in blood perfusion of the CL may not be a primary mechanism involved in decreased reproductive efficiency of cattle during fescue toxicosis.