Trans-10, cis-12 conjugated linoleic acid and the PPAR-γ agonist rosiglitazone attenuate lipopolysaccharide-induced TNF-α production by bovine immune cells.

Lipopolysaccharide (LPS) modulates innate immunity through alteration of cytokine production by immune cells. The objective of this study was to examine the effect of exogenous conjugated linoleic acid (CLA) and PPAR-γ agonist, rosiglitazone, on LPS-induced tumor necrosis factor α (TNF-α) production by cultured whole blood from prepubertal Holstein heifers (mean age, 5.5 mo). Compared with unstimulated cells, addition of LPS (10 µg/mL) to the culture medium increased (P<0.03) peripheral blood mononuclear cell proliferation≤2.5-fold. Coincubation with interferon γ (5 ng/mL) further stimulated (P<0.01) the lymphoproliferative response to LPS. Lipopolysaccharide increased (P<0.01) TNF-α concentration in cultured whole blood in a dose- and time-dependent manner. The greatest TNF-α stimulation occurred after 12 h of exposure to 1 µg/mL LPS. Coincubation with trans-10, cis-12 CLA isomer (100 µM) or rosiglitazone (10 µM), a PPAR-γ agonist, decreased (P<0.01) LPS-induced TNF-α production by 13% and 29%, respectively. Linoleic acid and cis-9, trans-11 CLA isomer had no detectable effects on LPS-induced TNF-α production in cultured bovine blood. The PPAR-γ agonist-induced TNF-α attenuation was reversed when blood was treated with both rosiglitazone and GW9662, a selective PPAR-γ antagonist. Addition of rosiglitazone to the culture medium tended to reduce nuclear factor-κ Bp65 concentration in nuclear and cytosolic extracts isolated from cultured peripheral blood mononuclear cells. Results show that LPS is a potent inducer of TNF-α production in bovine blood cells and that trans-10, cis-12 CLA and PPAR-γ agonists may attenuate the pro-inflammatory response induced by LPS in growing dairy heifers. Additional studies are needed to fully characterize the involvement of nuclear factor-κ B in LPS signaling in bovine blood cells.

Performance, metabolic, and endocrine responses of periparturient Holstein cows fed 3 sources of fat.

The objective of this study was to examine the effect of feeding diets containing fat supplements enriched in either saturated fatty acids (n = 10), Ca salts of trans-octadecenoic fatty acids (tFA, n = 10) or Ca salts of safflower oil fatty acids (SFL, high in linoleic acid, n = 9) on performance, metabolic, and endocrine responses of periparturient Holstein cows. Dietary treatments were initiated at approximately 28 d before calculated calving dates and continued through 49 d postpartum. Blood samples for metabolite and hormone analyses were collected weekly beginning 1 wk before estimated calving date through 7 wk postpartum. Incorporation of tFA or SFL into the peripartum diet had no detectable effects on body weight or body condition score. Cows fed the SFL-enriched diet produced less milk fat and established a positive energy balance sooner after calving than those fed the tFA supplement. Analysis for individual fatty acids resulted in increased concentrations of trans 18:1 fatty acid and conjugated linoleic acid isomers in milk fat from cows supplemented with SFL. Across weeks, the average nonesterified fatty acids concentration in plasma was lower in cows fed the SFL-enriched diet than in those consuming the tFA-supplemented diet. Mean concentrations of plasma glucose, insulin-like growth factor-I, and progesterone were greater in cows fed the SFL-enriched diet compared with those fed the saturated fatty acid-supplemented diet. Feeding fat supplements that can suppress milk fat production during the early postpartum period may help minimize negative energy balance, reduce adipose tissue mobilization, and improve circulating concentrations of insulin-like growth factor-I and progesterone. Whether the SFL supplement would have similar effects without a decrease in milk fat production remains to be determined and warrants further investigation.

Plasma prostaglandin and cytokine concentrations in periparturient Holstein cows fed diets enriched in saturated or trans fatty acids.

After parturition, immune functions such as lymphocyte response to mitogens and production of antibodies are depressed in dairy cows. Dietary regimens that improve the immune function of dairy cows after calving may improve uterine health and lead to earlier breeding after parturition. The objective of this study was to examine the effect of feeding a calcium salt of trans isomers of fatty acids (tFA) to periparturient Holstein cows on plasma biomarkers of inflammation. Dietary treatments were initiated approximately 28 d before expected calving date and continued through d 21 postpartum. Prepartum and postpartum diets were formulated to be isolipidic, containing 1.5% saturated fats (n = 15) or 1.8% tFA (n = 15). Multiparous cows were heavier at calving (+32%) and produced more milk (+17%) than primiparous cows. Periparturient tFA supplementation increased plasma PGF(2alpha) metabolite concentration in multiparous cows, but not in primiparous cows. Concentrations of prostaglandin E(2), tumor necrosis factor-alpha, and interleukin-4 in plasma did not differ between diets and parities. Results raise the possibility that peripartum tFA supplementation may affect uterine health and reproductive efficiency of early lactation dairy cows through alteration of peripheral PGF(2alpha) concentration.

Effects of dietary n-6:n-3 fatty acid ratio on feed intake, digestibility, and fatty acid profiles of the ruminal contents, liver, and muscle of growing lambs.

This study investigated the effect of modifying the n-6:n-3 fatty acid ratio (FAR) of diets using linseed, soybean, and cottonseed oils on apparent digestibility, ruminal fermentation characteristics, growth performance, key circulating hormones, and the fatty acid profile of ruminal digesta, liver, and fore-shank muscle of growing lambs fed a high concentrate diet. Forty individually housed Katadhin Dorper lambs (average of 20.0 kg of BW) were fed Bermudagrass hay in ad libitum amounts and concentrates at 3.7% of BW daily. The concentrate contained 68.9% corn, 23.8% soybean meal, 3.3% limestone, and 4.0% oil supplements (DM basis). The treatments consisted of dietary n-6:n-3 FAR of 2.3:1, 8.8:1, 12.8:1, and 15.6:1. After feeding for 35 d in metabolism crates, lambs were slaughtered 15 h after feeding, and samples of ruminal digesta, blood, liver, and foreshank tissue were collected. Increasing dietary n-6:n-3 FAR did not affect the intake of DM nor the apparent digestibility of DM, ether extract, NDF, or ADF, but did increase apparent digestibility of CP (linear, P < 0.05). Concentrations of ruminal butyrate increased linearly (P < 0.05) with increasing dietary n-6:n-3 FAR, whereas the valerate concentration decreased linearly (P < 0.001). Concentrations of plasma insulin and IGF-I were not affected by dietary n-6:n-3 FAR. Concentrations of C18:3n-3 increased linearly (P < 0.001), whereas that of C18:2n-6 decreased linearly (P < 0.001) in ruminal digesta with decreasing dietary n-6:n-3 FAR. Concentrations of transisomers of fatty acids in ruminal digesta did not change. Proportions of C18:0 in liver and foreshank muscle were unchanged by diet. The proportion of trans11 C18:1 and cis-9 trans11 CLA decreased (P < 0.05) in liver but increased (P < 0.05) in foreshank muscle as dietary n-6:n-3 FAR decreased. Proportions of all measured n-3 fatty acids were greater in liver when diets contained more C18:3n-3 from linseed oil. By decreasing the dietary n-6:n-3 FAR, the proportions of n-6 fatty acids in foreshank muscle decreased dramatically; specifically, C18:2n-6 decreased linearly (P < 0.001) from 28.0 to 16.5% and C20:4n-6 decreased linearly (P < 0.001) from 14.7 to 8.6%. Although feeding a diet that contained more n-3 fatty acids increased the n-3 fatty acid concentration of muscle, the ratio of PUFA to SFA was decreased.

Conjugated linoleic acid reduces phorbol ester-induced prostaglandin F2alpha production by bovine endometrial cells.

Recent interest in conjugated linoleic acid (CLA) research stems from the well-documented anticarcinogenic, antiatherogenic, antidiabetic, and antiobesity properties of CLA in animal models. The objective of this study was to examine the effects of 2 CLA isomers (cis-9,trans-11 and trans-10,cis-12) on phorbol 12,13-dibutyrate (PDBu)-induced PGF2alpha production in cultured bovine endometrial (BEND) cells. Confluent BEND cells were incubated in the absence (control) or presence of 100 microM each of linoleic acid, cis-9,trans-11 CLA, or trans-10,cis-12 CLA for 24 h. After incubation, cells were rinsed and then stimulated with PDBu (100 ng/mL) for 6 h. Compared with untreated cells, PDBu stimulated PGF2alpha secretion (+25-fold) within 6 h. The increases in PGF(2alpha) secretion were paralleled by signifi-cant induction of prostaglandin endoperoxide synthase-2 (PGHS-2) mRNA (+63-fold) and protein (+1.6-fold) expression. In spite of stimulatory effects on PGHS-2 and peroxisome proliferator-activated receptor delta (PPARdelta) mRNA responses, CLA greatly decreased PGF2alpha production by PDBu-stimulated BEND cells. There was no evidence for PDBu or CLA modulation of PPARdelta protein synthesis in cultured BEND cells. Results indicated that CLA modulation of PGF2alpha production by BEND cells was not mediated through PGHS-2 or PPARdelta gene repression.

Differential effects of n-3 and n-6 fatty acids on prostaglandin F2alpha production by bovine endometrial cells.

Recent studies have implicated n-3 polyunsaturated fatty acids in the reduction of eicosanoid production in the bovine uterus. The objective of this study was to determine whether the effect of eicosapentaenoic acid (EPA; C(20:5), n-3) on PGF2alpha production by bovine endometrial (BEND) cells is influenced by the quantity of linoleic acid (C(18:2), n-6) in the incubation medium. Confluent BEND cells were incubated in the absence (control) or presence of 100 microM of EPA for 24 h. After incubation, cells were rinsed and then stimulated with phorbol 12,13-dibutyrate (PDBu; 100 ng/mL) for 6 h. Additional sets of culture dishes were treated with a combination of EPA and increasing n-6/n-3 fatty acid ratios for 24 h and then challenged with PDBu for 6 h. The PDBu stimulated PGF2alpha secretion and upregulated steady-state concentrations of prostaglandin endoperoxide synthase-2 and peroxisome proliferator-activated receptor delta mRNA within 6 h. Preincubation of BEND cells with EPA for 24 h decreased PGF2alpha response to phorbol ester, but had no detectable effects on prostaglandin endoperoxide synthase-2 or peroxisome proliferator-activated receptor delta mRNA abundance in PDBu-stimulated BEND cells. The inhibitory effect of EPA on PGF2alpha production was reverted in BEND cells treated with an increasing n-6-to-n-3 fatty acid ratio. Findings indicate that the net inhibition of endometrial PGF2alpha bioynthesis by n-3 fatty acids may vary depending on the ratio of n-6 to n-3 fatty acids in the uterus.

Effects of bovine somatotropin on uterine genes related to the prostaglandin cascade in lactating dairy cows.

Multiparous Holstein cows, averaging 80 d in milk, were used to examine the effect of exogenous bovine somatotropin (bST) on uterine expression of estrogen receptor alpha (ERalpha), prostaglandin endoperoxide synthase-2 (PGHS-2), and peroxisome proliferator-activated receptor delta (PPARdelta). About 12 h before expected ovulation in a synchronization protocol, cows were assigned to receive bST (500 mg, n = 11) or serve as untreated controls (n = 10). Cows that ovulated (n = 9 bST, 8 control) were divided within treatment to be killed on d 3 or 7 postovulation. Samples of intercaruncular endometrial tissue from uterine horns ipsilateral to the corpus luteum were collected and stored at -80 degrees C for subsequent mRNA analyses. Endometrial concentrations of ERalpha and PGHS-2 mRNA transcripts were greater on d 7 than on d 3 of the estrous cycle, but did not differ between treatments. Compared with untreated cows, short-term bST treatment decreased PGHS-2 protein expression at d 7 of the estrous cycle. Concentration of PPARdelta mRNA transcript in the uterus decreased between d 3 and 7 of the estrous cycle and was negatively correlated with ERalpha and PGHS-2 mRNA concentrations. Short-term administration of bST to lactating dairy cows had minimal effects on uterine genes encoding ERalpha, PGHS-2, and PPARdelta at d 3 and 7 of the estrous cycle but there may be an inverse relationship between PPARdelta and uterine expression of ERalpha and PGHS-2 genes.

Inhibition of phorbol ester-induced PGF2alpha secretion by IFN-tau is not through regulation of protein kinase C.

Inhibitory effect of IFN-tau on phorbol ester (PdBu)-induced PGF2alpha secretion was hypothesized to be manifested by the regulation of protein kinase C (PKC) in bovine endometrial (BEND) cells. Following 12 h stimulation with PdBu, cells were unresponsive to freshly added PdBu. Pretreatment of cells with a PKC inhibitor abolished PGF2alpha secretion in response to PdBu. Therefore, PdBu induction of PGF2alpha secretion is through activation of PKC. The alpha, epsilon, iota and lambda isotypes of the PKC family were identified by Western blotting. Cells were then treated with medium alone (control), PdBu or PdBu + IFN-tau for 3 or 6 h. The PdBu-induced secretion of PGF2alpha was suppressed by IFN-tau. At 3 and 6 h, PKCalpha and PKCepsilon were detected both in the cytosolic and membrane fractions of unstimulated cells. There was a clear reduction of PKCalpha in the cytoplasm induced by PdBu and PdBu + IFN-tau at 3 and 6 h. The total abundance (cytoplasm and membrane fractions) of PKCalpha was lower in the PdBu + IFN-tau than PdBu alone. These temporal responses indicate a PKCalpha responsiveness of BEND cells to PdBu and PDBu + INF-tau with some evidence that IFN-tau causes a slight but detectable reduction in PKCalpha when added with PdBu. However, IFN-tau-induced decrease in the total abundance of PKCalpha was not enough to affect negatively the translocation of the PKCalpha to the membrane. Therefore, IFN-tau's ability to suppress secretion of PGF2alpha is unlikely due to an interference with the PdBu-induced activity of PKC.

Effects of biotin supplementation on peripartum performance and metabolites of Holstein cows.

Fifty-two multiparous Holstein cows were randomly assigned to receive 0 or 20 mg of biotin/d starting at an average of 16 d prepartum and then switched to 0 or 30 mg of biotin/d from calving through 70 d postpartum to determine whether supplemental biotin would affect cow performance, hepatic lipidosis, and plasma metabolites. Mean concentration of biotin in plasma sampled weekly was greater in cows fed biotin (4.3 vs. 9.4 nmol/L). Postpartum dry matter intake as a percentage of body weight (3.9% vs. 4.0%), milk production (35.8 vs. 34.8 kg/d), and milk fat concentrations (3.59% vs. 3.69%) were similar between treatment groups. Milk from biotin-supplemented cows tended to have a greater concentration of protein (2.73% vs. 2.83%). Concentrations of plasma nonesterified fatty acids were lower at wk 2 (652 vs. 413 microEq/mL) and 4 (381 vs. 196 microEq/mL) postpartum in cows fed supplemental biotin. However, mean plasma concentrations of beta-hydroxybutyric acid were not affected by biotin supplementation. Mean concentration of plasma glucose was greater for lactating cows fed supplemental biotin (63.4 vs. 66.6 mg/dL). Biopsies of liver were taken at 2, 16, and 30 d postpartum. The triacylglycerol concentration in liver (wet basis) tended to decrease at a faster rate after d 2 postpartum with biotin supplementation compared with control cows. The potential mechanisms that link improved glucose status and decreased lipid mobilization in cows supplemented with biotin warrant further investigation.

Production and metabolic responses of periparturient Holstein cows to dietary conjugated linoleic acid and trans-octadecenoic acids.

Thirty-eight multiparous Holstein cows were utilized in a completely randomized design to examine the effect of feeding calcium salts of conjugated linoleic acid (CLA) and trans-octadecenoic acids (trans-C18:1) on animal performance and lipid and glucose metabolism during the transition to lactation. Dietary treatments were initiated approximately 28 d prior to expected calving dates and continued through d 49 postpartum. Prepartum treatments consisted of 1) a basal diet (Control), 2) basal diet + 150 g/d of CLA mix (CLA), and 3) basal diet + 150 g/d of trans-C18:1 mix (TRANS). Amounts of calcium salts of CLA and trans-C18:1 mixes were adjusted to 225 g/d during the 49-d postpartum treatment period. All diets were offered as a total mixed ration. Prepartum fat supplementation had no detectable effects on dry matter intake, body weight, or body condition score. After parturition, cows in the TRANS group consumed less dry matter at wk 4, 5, and 6 of lactation than did cows in the control group. Cows fed the trans-C18:1 supplement were in a more severe negative energy balance than those fed the control diet at 1 wk of lactation. Periparturient fat supplementation had no detectable effects on milk yield during wk 1 to 7 of lactation. Milk fat was not affected during wk 1 to 4, but was reduced after wk 4 of lactation by dietary CLA. Feeding calcium salts of CLA decreased short- to medium-chain fatty acid (C4 to C14) concentrations and increased both linoleic and linolenic acid concentrations in milk fat. Concentrations of nonesterified fatty acids and beta-hydroxybutyric acid in blood were greater in cows fed the CLA-supplemented diet than in those fed the control diet at 1 wk of lactation. In spite of small numerical tendencies, hepatic lipid and triacylglycerol concentrations did not vary significantly among dietary treatments. Periparturient fat supplementation had no detectable effects on plasma glucose and insulin concentrations. Steady-state concentrations of hepatic mRNA encoding pyruvate carboxylase and phosphoenolpyruvate carboxykinase were greater for the TRANS treatment group than the control and CLA groups. Results indicate that dietary CLA and trans-C18:1 fatty acids may affect lipid and glucose metabolism in early postpartum Holstein cows through distinct mechanisms.

Regulation of embryo survival in cattle.

Evidence is presented that bovine somatotrophin (bST) treatment of lactating dairy cows enhances both expression of oviductal insulin-like growth factor II (IGF-II) mRNA and endometrial insulin-like growth factor binding protein 3 (IGFBP-3) mRNA between day 3 and day 7 of the oestrous cycle. mRNA encoding growth hormone (GH) receptor in endometrial tissues increased between day 3 and day 7 of the oestrous cycle. The changes induced by bST treatment may contribute to stimulation of embryo development and increase pregnancy rates in lactating dairy cows. Additive effects of bST and rb interferon tau (rbIFN-tau) to inhibit phorbol ester induction of prostaglandin F2alpha secretion in immortalized bovine endometrial cells indicates that there is interplay between their signal transduction pathways. Non-lactating dairy cows were killed at day 17 after oestrus to evaluate the effects of pregnancy status (cyclic versus pregnant) and bST (bST versus control) treatment on endometrial gene expression. Distinctly different mRNA and protein responses were detected between cyclic and pregnant cows that were related to luteolytic-antiluteolytic drive (that is expression of progesterone receptor, oxytocin receptor, oestradiol receptor alpha and prostaglandin GH synthase 2 (PGHS-2)). The bST-induced changes in PGHS-2 protein (+), oxytocin receptor mRNA (+) and oestrogen receptor alpha protein (+) may potentially affect the mechanisms associated with maintenance of pregnancy. Two experiments were conducted to evaluate whether ovarian follicular suppression induced by biodegradable deslorelin implants would reduce either early or late embryo losses. A 450 microg deslorelin implant used to induce ovulation in a timed insemination programme decreased subsequent follicular development and tended to reduce early embryo losses, whereas a 2.1 mg deslorelin implant failed to reduce late embryonic losses when inserted on day 27 of pregnancy.

Dietary conjugated linoleic acid alters hepatic lipid content and fatty acid composition in broiler chickens.

We examined the effect of dietary conjugated linoleic acid (CLA) on growth performance and liver composition in broiler chickens. Day-old male broiler chicks were assigned to receive a diet supplemented with corn oil (5%; n = 48) or CLA (5%; n = 48) for 21 d. Broilers fed CLA weighed less and grew at slower rates than broilers fed corn oil. Feed intake and feed conversion were higher for the corn-oil group than for the CLA dietary group. Hepatic lipid and triacylglycerol concentrations were significantly reduced by dietary CLA. The proportions of saturated fatty acids (SFA) in liver lipids increased, whereas those of monounsaturated fatty acids (MUFA) decreased with CLA supplementation. Although the total concentration of polyunsaturated fatty acids (PUFA) did not change with dietary treatment, the concentration of linoleic acid as a percentage of total methylated fatty acids decreased, and that of linolenic add increased in broilers fed CLA. The concentration of CLA isomers in liver lipids increased substantially with CLA feeding. The relative proportion of the c9,t11 CLA isomer in hepatic lipids was much higher than that of the t10,c12 or t9,t11 CLA isomers. These studies provide evidence that feeding CLA to broilers results in substantial reduction in liver fat accumulation and promotes CLA incorporation into hepatic lipid pools.

Effects of growth hormone and insulin-like growth factor-I on development of in vitro derived bovine embryos.

The objectives of this study were to determine whether the addition of growth hormone (GH) to maturation medium and GH or insulin-like growth factor-I (IGF-I) to culture medium affects development of cultured bovine embryos. We matured groups of 10 cumulus-oocyte complexes (COCs) in serum-free TCM-199 medium containing FSH and estradiol with or without 100 ng/ml GH. After fertilization, we transferred groups of 10 putative zygotes to 25 microl drops of a modified KSOM medium containing the following treatments: non-specific IgG (a control antibody, 10 microg/ml); GH (100 ng/ml) + IgG (10 microg/ml, GH/IgG); IGF-I (100 ng/ml) + IgG (10 microg/ml, IGF/IgG); antibody to IGF-I (10 microg/ml, anti-IGF); GH (100 ng/ml) + anti-IGF (10 microg/ml GH/anti-IGF); IGF-I (100 ng/ml) + anti-IGF (10 microg/ml, IGF/anti-IGF); no further additions (control). We repeated the experiment six times. Adding GH to the maturation medium increased cleavage rates at Day 3 compared to control (87.3 +/- 1.2% > 83.9 +/- 1.2%; P < 0.05) but had no effects on blastocyst development at Day 8. At Day 8, blastocyst development was greater (P < 0.01) for GH/IgG (24.8 +/- 2.5%) and IGF/IgG (33.7 +/- 2.5%) than for IgG (16.1 +/- 2.1%) and greater for IGF/IgG than for GH/IgG (P < 0.02). Blastocyst development at Day 8 did not differ between anti-IGF (20.4 +/- 1.8%) and GH/anti-IGF (24.1 +/- 1.9%) or IGF/anti-IGF (17.7 +/- 1.9%), but it was greater for GH/anti-IGF than for IGF/anti-IGF (P < 0.05). The Day 8 blastocysts of GH/IgG and IGF-I/IgG groups had a higher (P < 0.01) number of cells than the IgG group. The addition of anti-IGF-I eliminated the effects of IGF-I on cell number but did not alter GH effects. In conclusion, both GH and IGF-I stimulate embryonic development in cattle and GH effects may likely involve IGF-I-independent mechanisms.

Bovine somatotropin increases embryonic development in superovulated cows and improves post-transfer pregnancy rates when given to lactating recipient cows.

Previous studies indicated that the use of bovine somatotropin (bST) in concurrence with a timed artificial insemination (TAI) protocol increased pregnancy rates. However, the mechanisms for such a bST effect on fertility were not clear. Objectives of this study were to determine the effects of bST on fertilization and early embryonic development after cows received a superovulation treatment, test whether embryos recovered from bST-treated cows were more likely to survive after transfer to recipients, and evaluate whether treatment of recipient cows with bST affects pregnancy rates. Lactating (n = 8) and nonlactating (n = 4) Holstein donor cows were superovulated, inseminated at detected estrus and assigned to a nontreated control group or to a treatment group receiving a single injection of bST (500 mg, sc) at insemination. Embryos were nonsurgically flushed 7 days after AI and frozen in ethylene glycol for direct transfer. Embryos derived from bST-treated (bST-embryos) or control (control-embryos) donors were transferred to lactating Holstein recipient cows that received either bST treatment 1 day after estrus (500 mg, sc; bST-recipients) or were untreated controls (control-recipients). Thus, there were four treatment groups: control-embryos/control-recipients (n = 43), bST-embryos/control-recipients (n = 41), control-embryos/bST-recipients (n = 37), and bST-embryos/bST-recipients (n = 60). Pregnancy was determined by palpation per rectum 33-43 days after embryo transfer. Unfertilized ova per flush was less for bST than for control (1.0 +/- 0.9 < 3.7 +/- 0.9; P < 0.04). Percentage of transferable embryos was greater for bST than for control (77.2% > 56.4%; P < 0.01). Number of blastocysts per flush was greater for bST than for control (2.4 +/- 0.7 > 0.4 +/- 0.7; P < 0.04). Pregnancy rates following embryo transfer were 25.6% for control-recipient/control-embryo, 43.2% for bST-recipient/control-embryo, 56.1% for control-recipient/bST-embryo, and 43.3% for bST-recipient/bST-embryo. Transfer of bST-embryos increased pregnancy rates compared with transfer of control-embryos (P < 0.04). An interaction between embryo and recipient treatments (P < 0.05) indicated that treatment of recipient cows with bST increased pregnancy rates as compared to control-recipients that received a control-embryo. However, there was no additive effect when bST-recipients received a bST-embryo. Administration of bST at AI decreased the number of unfertilized ova, increased the percentage of transferable embryos, and stimulated embryonic development to the blastocyst stage. Moreover, bST affected both early embryonic development and recipient components to increase pregnancy rates following embryo transfer.

Short communication: Hepatic gene expression for gluconeogenic enzymes in lactating dairy cows treated with bovine somatotropin.

Eight lactating Holstein dairy cows (80 d in milk) were used to examine the effects of exogenous bovine somatotropin (bST) on hepatic contents of mRNA encoding pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), and microsomal triglyceride transfer protein (MTP). Concentrations of bST in plasma were higher and milk production increased 20% in bST-treated cows. Liver samples from cows treated with bST had significantly higher total lipid contents than those from control cows. Although there were small numerical tendencies, neither triglyceride concentrations in liver nor nonesterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA), or glucose in plasma differed significantly between bST-treated and control cows. Short-term bST treatment had no detectable effects on contents of PC, PEPCK, and MTP mRNA in the liver. In summary, exogenous bST stimulation of milk production is not mediated through enhanced liver gluconeogenesis, but may involve partitioning of glucose and fatty acids for preferential use by the mammary gland.

Bovine somatotropin attenuates phorbol ester-induced prostaglandin F2alpha production in bovine endometrial cells.

The recent observation that bovine somatotropin (bST) treatment at a timed insemination improves pregnancy rates in lactating dairy cows raises the possibility that growth hormone (GH) may modulate the endocrine and biochemical cross talk between the conceptus and maternal uterus at the time of pregnancy establishment in cattle. The objective of this study was to characterize the cellular and molecular mechanisms by which exogenous GH affects phorbol ester-induced prostaglandin F2alpha (PGF2alpha) production in cultured bovine endometrial (BEND) cells. Serum-deprived BEND cells were incubated with or without recombinant bovine GH (rbGH), insulin-like growth factor (IGF)-I, recombinant bovine interferon (rbIFN)-tau or a combination of rbGH + rbIFN-tau for 3 h and then treated with phorbol 12,13-dibutyrate (PDBu) for an additional 6 h. Exogenous PDBu increased PGF2alpha secretion and steady-state levels of COX-2 mRNA within 3 h. Priming of BEND cells with rbGH reduced PGF2alpha response to PDBu, whereas cotreatment with IGF-I amplified PDBu induction of PGF2alpha. Preincubation of cell monolayers with rbIFN-tau suppressed PGF2alpha and COX-2 mRNA responses to PDBu. Inhibitory effects of rbGH and rbIFN-tau on PDBu-induced PGF2alpha production were additive. Results provide the first direct evidence that supplemental bST may interact with conceptus-secreted IFN-tau to modulate PGF2alpha secretion at the critical time of maternal recognition of pregnancy.

Effects of BST on oviductal and uterine genes encoding components of the IGF system in lactating dairy cows.

Lactating Holstein cows, averaging 80 d in milk, were used to examine effects of exogenous bovine somatotropin (bST) on oviductal and uterine genes encoding components of the insulin-like growth factor (IGF) system. About 12 h before expected ovulation in an Ovsynch protocol, cows were assigned randomly to receive bST (500 mg; n = 11) or serve as untreated controls (n = 10). Cows that ovulated (n = 9 bST, 8 control) were divided within treatment to be sacrificed on d 3 or 7 postovulation. Samples of oviductal and intercaruncular endometrial tissue from oviducts and uterine horns ipsilateral to the corpus luteum (CL) were collected and immediately frozen at -80 degrees C for subsequent mRNA analyses. Northern blots revealed mRNAs for IGF-II, IGF-binding protein-2 (IGFBP-2), and IGFBP-3 in all oviductal and endometrial tissues. Significant amounts of IGF-I and growth hormone receptor-1A (GHR-1A) mRNAs were detected in uteri but not in oviducts. The bST treatment had no effect on amount of IGF-I mRNA transcript in uterine endometrium. The mRNA encoding IGF-II was induced by bST in oviducts collected on both d 3 and 7 but was down-regulated in endometrium on d 7. Transcript of IGFBP-2 mRNA was greater in endometrial than oviductal tissues and did not differ between treatments. Both oviductal and endometrial IGFBP-3 mRNA concentrations increased between d 3 and 7 postovulation, with a tendency for greater endometrial IGFBP-3 mRNA in bST-treated cows on d 7. On d 7, concentrations of endometrial GHR-1A mRNA were 30% lower in bST-treated cows. Results indicate complex and tissue-specific regulation of the uterine IGF system components by exogenous bST. Some of those biological responses to bST may be important in early development of bovine embryos.

Bovine interferon-tau stimulates the Janus kinase-signal transducer and activator of transcription pathway in bovine endometrial epithelial cells.

Trophoblastic bovine interferon-tau (bIFN-tau) suppresses luteolytic pulses of endometrial prostaglandin F(2alpha) (PGF(2alpha)) at the time of maternal recognition of pregnancy. This results in maintenance of the corpus luteum in cattle. The hypothesis that effects of bIFN-tau in the endometrium were through activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway of signal transduction was tested. Whole cell, cytosolic, and nuclear extracts from bovine endometrial cells treated with bIFN-tau were analyzed by immunoprecipitation, immunoblotting, and electrophoretic mobility shift assays in a series of dose- and time-dependency experiments. Bovine IFN-tau stimulated tyrosine phosphorylation, homo- and heterodimer formation, nuclear translocation, and DNA binding of STAT proteins 1, 2, and 3. Moreover, bIFN-tau induced synthesis of interferon-regulatory factor. In conclusion, bIFN-tau stimulates the JAK-STAT pathway in the bovine endometrium. It is proposed that activation of the JAK-STAT pathway is involved in regulating the antiluteolytic effects of bIFN-tau.

Interferon-tau modulates phorbol ester-induced production of prostaglandin and expression of cyclooxygenase-2 and phospholipase-A(2) from bovine endometrial cells.

Antiluteolytic actions of bovine interferon-tau (bIFN-tau) require suppression of prostaglandin F(2 alpha) (PGF(2 alpha)) production. Our objective was to test whether bIFN-tau could block PGF(2 alpha) production and synthesis of phospholipase A(2) (PLA(2)) and cyclooxygenase-2 (COX-2) enzymes induced by a protein kinase C (PKC) stimulator (phorbol 12,13 dibutyrate; PDBu). Bovine endometrial epithelial (BEND) cells were treated with PDBu in the presence or absence of bIFN-tau. Medium samples were analyzed for concentrations of PGF(2 alpha), whole-cell extracts were analyzed for abundance of PLA(2) and COX-2 by immunoblotting, and RNA extracts were examined for steady-state levels of COX-2 mRNA by Northern blotting. The PDBu stimulated production of PGF(2 alpha) between 3 and 12 h, levels of COX-2 mRNA by 3 h and protein expression of COX-2 and PLA(2) by 6 and 12 h, respectively. Added concomitantly with PDBu, bIFN-tau suppressed PGF(2 alpha) production, steady-state levels of COX-2 mRNA, and expression of COX-2 and PLA(2) proteins. Added after a 3-h stimulation with PDBu alone, bIFN-tau suppressed PGF(2 alpha) production after 1 h. Bovine IFN-tau inhibited intracellular mechanisms responsible for PGF(2 alpha) production in BEND cells, and this could be through both cytosolic and nuclear actions.

Complex mediation of uterine endometrial epithelial cell growth by insulin-like growth factor-II (IGF-II) and IGF-binding protein-2.

The coexpression of IGF (-I and -II) peptides, corresponding receptors, and IGF binding proteins (IGFBPs) in uterine endometrium suggests that a significant component of IGF action in this tissue is via autocrine or paracrine pathways, or both. The present study examined whether IGF-II and a major uterine-expressed IGF-II binding protein, IGFBP-2, modulate endometrial epithelial cell mitogenesis. Serum-deprived porcine endometrial glandular epithelial (GE) cells of early pregnancy were treated with various concentrations of IGFs, recombinant porcine (rp) IGFBP-2, or both, and examined for changes in cellular mitogenesis by incorporation of [(3)H]thymidine into DNA. Recombinant human (rh) IGF-II stimulated DNA synthesis in a dose-dependent manner. Human [Leu(27)]-IGF-II, an analog with selective affinity for the IGF-II (type II) receptor, increased thymidine uptake by twofold compared with untreated GE cells. When added in combination with an equimolar concentration of rhIGF-I, [Leu(27)]-IGF-II or rhIGF-II stimulated thymidine incorporation to a greater extent than did rhIGF-I alone. Ligand blot analysis of GE cell conditioned medium revealed the presence of four IGFBPs with molecular masses of 48, 31, 23, and 15 kDa. Physiological concentrations of rpIGFBP-2 (nM range) increased both basal and IGF-induced DNA synthesis in GE cells. At equimolar concentrations, Des(1-6)IGF-II (an IGF-II analog with much reduced affinity for IGFBPs) and rpIGFBP-2 had additive effects on GE cell mitogenesis, suggesting that the IGFBP-2 modulation of uterine cell growth may involve both IGF-dependent and IGF-independent pathways. Our results demonstrate the complex interplay of IGF system components in uterine endometrial epithelial growth regulation in vitro, identify IGF-II and IGFBP-2 as locally coexpressed uterine epithelial cell mitogens, and suggest the presence of a functional signaling pathway by which IGF-II stimulates epithelial cell proliferation via the type II receptor.