Effects of abomasal infusion of nicotinic acid on responses to glucose and ß-agonist challenges in underfed lactating cows.

The objectives were to assess the use of nicotinic acid (NA) to chronically (i.e., 74 h) manipulate plasma nonesterified fatty acid (NEFA) concentrations in partially feed-restricted lactating cows, determine whether the reduction of plasma NEFA altered responses to i.v. glucose tolerance test (ivGTT) and whether NA would attenuate an acute lipolytic stimuli of a ß-agonist challenge (ivBAC). Eight lactating dairy cows [244 ± 31 d in milk; 696 ± 63 kg of body weight (BW)] were blocked by breed and body condition score (3.2 ± 0.4) and randomly assigned to a sequence of 2 treatments in a crossover design. Treatments were 74-h continuous abomasal infusion of NA solution (3mg/h per kg of BW) as an antilipolytic agent to decrease plasma NEFA concentrations or the same volume of water (200 mL/h), concomitant with partial feed restriction. From 0 to 74 h of each experimental period, cows were feed-restricted to 33% of the ad libitum intake recorded during the prior 5 d. An ivGTT (0.25 g/kg of BW of glucose i.v.) and an ivBAC (4 nmol/kg of BW of isoproterenol hydrochloride, i.v.) were performed at 48 and 72 h, respectively. Intake was 24.1, 8.2, 8.0, and 8.0 kg of dry matter/d before restriction, on d 1, 2 and 3, respectively. Nicotinic acid decreased plasma NEFA and increased insulin and glucose concentrations during feed restriction. Nicotinic acid also led to greater glucose and insulin response areas under the curve during ivGTT [glucose: 6,562 vs. 5,056 (mg/dL) × 180 min; insulin: 6,042 vs. 2,502 (µIU/mL) × 180 min] and ivBAC [glucose: 535 vs. 240 (mg/dL) × 120 min; insulin: 1,283 vs. 222 (µIU/mL) × 120 min], and enhanced NEFA area under the curve during ivBAC [45,521 vs. 22,862 (µEq/L) × 120 min]. Milk, fat, and protein yields (29.1, 1.2, and 0.93 kg on d -2, respectively) decreased to 17.9, 0.81, and 0.56 kg for control, and 11.5, 0.54, and 0.39 kg for NA on d 3, respectively. Nicotinic acid may have decreased production by inhibiting the supply of NEFA for energy and milk fat synthesis. Milk urea nitrogen was increased by NA on d 2 (12.8 vs. 19.1mg/dL) and d 3 (11.6 vs. 17.8 mg/dL), probably due to a greater reliance on mobilized amino acids. Somatic cell count was increased by NA on d 3 (187 vs. 848 × 1,000 cells/mL). Patterns of glucose and insulin concentration observed during 74 h of NA infusion reflect a state of insulin resistance, which contrasts with shorter-term responses in nonlactating cows. Data suggest that long-term supraphysiological infusion of NA affected intermediary metabolism beyond antilipolysis and did not inhibit acute lipolytic stimuli of ivBAC.

Postovulatory aging affects dynamics of mRNA, expression and localization of maternal effect proteins, spindle integrity and pericentromeric proteins in mouse oocytes.

STUDY QUESTION: Is the postovulatory aging-dependent differential decrease of mRNAs and polyadenylation of mRNAs coded by maternal effect genes associated with altered abundance and distribution of maternal effect and RNA-binding proteins (MSY2)? SUMMARY ANSWER: Postovulatory aging results in differential reduction in abundance of maternal effect proteins, loss of RNA-binding proteins from specific cytoplasmic domains and critical alterations of pericentromeric proteins without globally affecting protein abundance. WHAT IS KNOWN ALREADY: Oocyte postovulatory aging is associated with differential alteration in polyadenylation and reduction in abundance of mRNAs coded by selected maternal effect genes. RNA-binding and -processing proteins are involved in storage, polyadenylation and degradation of mRNAs thus regulating stage-specific recruitment of maternal mRNAs, while chromosomal proteins that are stage-specifically expressed at pericentromeres, contribute to control of chromosome segregation and regulation of gene expression in the zygote. STUDY DESIGN, SIZE, DURATION: Germinal vesicle (GV) and metaphase II (MII) oocytes from sexually mature C57B1/6J female mice were investigated. Denuded in vivo or in vitro matured MII oocytes were postovulatory aged and analyzed by semiquantitative confocal microscopy for abundance and localization of polyadenylated RNAs, proteins of maternal effect genes (transcription activator BRG1 also known as ATP-dependent helicase SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) and NOD-like receptor family pyrin domain containing 5 (NLRP5) also known as MATER), RNA-binding proteins (MSY2 also known as germ cell-specific Y-box-binding protein, YBX2), and post-transcriptionally modified histones (trimethylated histone H3K9 and acetylated histone H4K12), as well as pericentromeric ATRX (alpha thalassemia/mental retardation syndrome X-linked, also termed ATP-dependent helicase ATRX or X-linked nuclear protein (XNP)). For proteome analysis five replicates of 30 mouse oocytes were analyzed by selected reaction monitoring (SRM). MATERIAL AND METHODS: GV and MII oocytes were obtained from large antral follicles or ampullae of sexually mature mice, respectively. Denuded MII oocytes were aged for 24 h post ovulation. For analysis of distribution and abundance of polyadenylated RNAs fixed oocytes were in situ hybridized to Cy5 labeled oligo(dT)20 nucleotides. Absolute quantification of protein concentration per oocyte of selected proteins was done by SRM proteome analysis. Relative abundance of ATRX was assessed by confocal laser scanning microscopy (CLSM) of whole mount formaldehyde fixed oocytes or after removal of zona and spreading. MSY2 protein distribution and abundance was studied in MII oocytes prior to, during and after exposure to nocodazole, or after aging for 2 h in presence of H2O2 or for 24 h in presence of a glutathione donor, glutathione ethylester (GEE). MAIN RESULTS AND ROLE OF CHANCE: The significant reduction in abundance of proteins (P < 0.001) translated from maternal mRNAs was independent of polyadenylation status, while their protein localization was not significantly changed by aging. Most of other proteins quantified by SRM analysis did not significantly change in abundance upon aging except MSY2 and GTSF1. MSY2 was enriched in the subcortical RNP domain (SCRD) and in the spindle chromosome complex (SCC) in a distinct pattern, right and left to the chromosomes. There was a significant loss of MSY2 from the SCRD (P < 0.001) and the spindle after postovulatory aging. Microtubule de- and repolymerization caused reversible loss of MSY2 spindle-association whereas H2O2 stress did not significantly decrease MSY2 abundance. Aging in presence of GEE decreased significantly (P < 0.05) the aging-related overall and cytoplasmic loss of MSY2. Postovulatory aging increased significantly spindle abnormalities, unaligned chromosomes, and abundance of acetylated histone H4K12, and decreased pericentromeric trimethylated histone H3K9 (all P < 0.001). Spreading revealed a highly significant increase in pericentromeric ATRX (P < 0.001) upon ageing. Thus, the significantly reduced abundance of MSY2 protein, especially at the SCRD and the spindle may disturb the spatial control and timely recruitment, deadenylation and degradation of developmentally important RNAs. An autonomous program of degradation appears to exist which transiently and specifically induces the loss and displacement of transcripts and specific maternal proteins independent of fertilization in aging oocytes and thereby can critically affect chromosome segregation and gene expression in the embryo after fertilization. LIMITATION, REASONS FOR CAUTION: We used the mouse oocyte to study processes associated with postovulatory aging, which may not entirely reflect processes in aging human oocytes. However, increases in spindle abnormalities, unaligned chromosomes and H4K12 acetylated histones, as well as in mRNA abundance and polyadenylation have been observed also in aged human oocytes suggesting conserved processes in aging. WIDER IMPLICATIONS OF THE FINDINGS: Postovulatory aging precociously induces alterations in expression and epigenetic modifications of chromatin by ATRX and in histone pattern in MII oocytes that normally occur after fertilization, possibly contributing to disturbances in the oocyte-to-embryo transition (OET) and the zygotic gene activation (ZGA). These observations in mouse oocytes are also relevant to explain disturbances and reduced developmental potential of aged human oocytes and caution to prevent oocyte aging in vivo and in vitro. STUDY FUNDING/COMPETING INTERESTS: The study has been supported by the German Research Foundation (DFG) (EI 199/7-1 | GR 1138/12-1 | HO 949/21-1 and FOR 1041). There is no competing interest.

Claudin-3, claudin-7, and claudin-10 show different distribution patterns during decidualization and trophoblast invasion in mouse and human.

Implantation of the mammalian embryo requires profound endometrial changes for successful pregnancy, including epithelial-mesenchymal transition of the luminal epithelium and stromal-epithelial transition of the stromal cells resulting in decidualization. Claudins (Cldn) determine the variability in tight junction paracellular permeability and may play a role during these epithelial and decidual changes. We here localized Cldn3, Cldn7 and Cldn10 proteins in the different compartments of murine endometrium up to day 8.5 of pregnancy (dpc) as well as in human endometrium and first trimester decidua. In murine estrous endometrium, luminal and glandular epithelium exhibited Cldn3 and Cldn7, whereas Cldn10 was only detectable in glandular epithelium. At 4.5 dpc, Cldn3 protein shifted to an apical localization, whereas Cldn7 vanished in the epithelium of the implantation chamber. At this stage, there was no stromal signal for Cldn3 and Cldn7, but a strong induction of Cldn10 in the primary decidual zone. Cldn3 proteins emerged at 5.5 dpc spreading considerably from 6.5 dpc onward in the endothelial cells of the decidual blood sinusoids and in the decidual cells of the compact antimesometrial region. In addition to Cldn3, Cldn10 was identified in human endometrial epithelia. Both proteins were not detected in human first trimester decidual cells. Cldn3 was shown in murine trophoblast giant cells as well as in human extravillous trophoblast cells and thus may have an impact on trophoblast invasion in both species. We here showed a specific claudin signature during early decidualization pointing to a role in decidual angiogenesis and regulation of trophoblast invasion.

Relationships between fertility and postpartum changes in body condition and body weight in lactating dairy cows.

The relationship between energy status and fertility in dairy cattle was retrospectively analyzed by comparing fertility with body condition score (BCS) near artificial insemination (AI; experiment 1), early postpartum changes in BCS (experiment 2), and postpartum changes in body weight (BW; experiment 3). To reduce the effect of cyclicity status, all cows were synchronized with Double-Ovsynch protocol before timed AI. In experiment 1, BCS of lactating dairy cows (n = 1,103) was evaluated near AI. Most cows (93%) were cycling at initiation of the breeding Ovsynch protocol (first GnRH injection). A lower percentage pregnant to AI (P/AI) was found in cows with lower (≤ 2.50) versus higher (≥ 2.75) BCS (40.4 vs. 49.2%). In experiment 2, lactating dairy cows on 2 commercial dairies (n = 1,887) were divided by BCS change from calving until the third week postpartum. Overall, P/AI at 70-d pregnancy diagnosis differed dramatically by BCS change and was least for cows that lost BCS, intermediate for cows that maintained BCS, and greatest for cows that gained BCS [22.8% (180/789), 36.0% (243/675), and 78.3% (331/423), respectively]. Surprisingly, a difference existed between farms with BCS change dramatically affecting P/AI on one farm and no effect on the other farm. In experiment 3, lactating dairy cows (n = 71) had BW measured weekly from the first to ninth week postpartum and then had superovulation induced using a modified Double-Ovsynch protocol. Cows were divided into quartiles (Q) by percentage of BW change (Q1 = least change; Q4 = most change) from calving until the third week postpartum. No effect was detected of quartile on number of ovulations, total embryos collected, or percentage of oocytes that were fertilized; however, the percentage of fertilized oocytes that were transferable embryos was greater for cows in Q1, Q2, and Q3 than Q4 (83.8, 75.2, 82.6, and 53.2%, respectively). In addition, percentage of degenerated embryos was least for cows in Q1, Q2, and Q3 and greatest for Q4 (9.6, 14.5, 12.6, and 35.2% respectively). In conclusion, for cows synchronized with a Double-Ovsynch protocol, an effect of low BCS (≤ 2.50) near AI on fertility was detected, but change in BCS during the first 3 wk postpartum had a more profound effect on P/AI to first timed AI. This effect could be partially explained by the reduction in embryo quality and increase in degenerate embryos byd 7 after AI in cows that lost more BW from the first to third week postpartum.

Effect of rumen-protected niacin on lipid metabolism, oxidative stress, and performance of transition dairy cows.

The objective of this study was to evaluate the effect of a rumen-protected niacin product (RPN; 65% nicotinic acid; NiaShure, Balchem Corp., New Hampton, NY) on lipid metabolism, oxidative stress, and performance of transition dairy cows. Thirty nonlactating multiparous Holstein cows in late gestation were paired according to expected calving date and randomly assigned to 12 g/cow per day of RPN product or to an unsupplemented control (CON) diet. Treatment diets were fed from 21 d before expected calving through 21 d after parturition. Blood samples were taken on d -21, -14, -7, 1, 7, 14, and 21 relative to calving for plasma nonesterified fatty acid (NEFA), ß-hydroxybutyrate (BHBA), glucose, and superoxide dismutase (SOD) analyses. Liver samples were taken by biopsy on d 1 and 21 relative to calving for triglyceride (TG) analysis. Data were analyzed for a randomized complete block design with repeated measures. Pre- and postpartum dry matter intake, milk yield, and protein were unaffected by treatment. Milk fat percentage (5.08 vs. 4.44%) and somatic cell score (3.93 vs. 2.48) were reduced for RPN. Treatment × time interactions were observed for energy-corrected milk (ECM) and fat-corrected milk (FCM) yields; RPN reduced ECM and FCM yields by 8.5 and 8.9 kg/cow per day, respectively, in the first week of lactation. Although body weight and condition score decreased during the experimental period, no differences due to treatment were observed. However, calculated postpartum energy balance tended to be improved for RPN because of the reduction in ECM yield. Time and treatment × time effects were observed for plasma NEFA. On d 1 postpartum, NEFA reached 1,138±80 µEq/L for CON compared with 698±80 µEq/L for RPN. Cows supplemented with RPN tended to have lower plasma NEFA concentrations than CON cows on d 7 and 14 postpartum. Plasma BHBA, glucose, and SOD and liver TG concentrations were unaffected by treatment. In conclusion, supplementation with 12 g/cow per day of the RPN product provided a bioavailable source of niacin that modified lipid metabolism but did not affect milk yield over the first 3 wk of lactation or oxidative stress of transition dairy cows.

Signalling of DNA damage and cytokines across cell barriers exposed to nanoparticles depends on barrier thickness.

The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.

Short communication: Effect of a stable pen management strategy for precalving cows on dry matter intake, plasma nonesterified fatty acid levels, and milk production.

During the close-up transition period, dairy cows are at risk for negative energy balance due to increasing energy demands and decreasing feed intake. This can result in postparturient health problems and decreased milk production after calving. Cows are frequently regrouped during this period, which can negatively affect feeding and resting behavior. The hypothesis was that housing in a stable pen during the close-up transition period should result in a more settled environment resulting in fewer displacements from the feed bunk, which would result in improved feed intake, energy balance [lower nonesterified fatty acid (NEFA) concentrations], and lactation performance. This study addresses precalving pen grouping strategies, which have the potential to affect feed intake and energy balance. A randomized complete block design with pen as the experimental unit was used to compare a stable (S) housing strategy (cows with similar calving dates added to a precalving pen at once) to the more traditional dynamic (D) housing strategy (cows added up to 2 times per week to a precalving pen). Twice-weekly blood samples were collected for NEFA analysis and cow interactions within the pen were observed. Dry matter intake (DMI), milk production, and postparturient health problems were recorded. Mean DMI for the duration of the 28 d of the study was not different (S: 25.5 ± 1.6 vs. D: 25.7 ± 1.0 kg/d), and when examined over time relative to calving, no treatment by time interaction was observed. Concentrations of NEFA were not different when cows initially entered the pens (S: 0.21 ± 0.10 vs. D: 0.18 ± 0.04 mEq/L) and remained not different for the time intervals closer to calving (d -9 to -14: S: 0.28 ± 0.09 vs. D: 0.21 ± 0.04; d -3 to -6: S 0.36 ± 0.10, D 0.32 ± 0.05 mEq/L). Pen grouping strategy did not affect DMI, plasma NEFA concentrations, or milk production.

Antilipolytic and lipolytic effects of administering free or ruminally protected nicotinic acid to feed-restricted Holstein cows.

The objectives were to determine effects of 12 hourly infusions of different quantities of nicotinic acid (NA) on plasma nonesterified fatty acid (NEFA; experiment 1) and whether longer (108 h) continuous infusions of NA could induce sustained reductions of plasma NEFA (experiment 2) in nonlactating, nongestating Holstein cows that were feed restricted. Experiment 1 was a 5×5 Latin square with 6-d periods and 9 recovery days between each period. Each period consisted of 5 d of partial feed restriction to increase plasma NEFA concentration. Treatments were abomasal infusions of 0, 0.25, 0.5, 1, or 3 mg of NA/h per kilogram of body weight (BW), infused as hourly boluses for 12 h, starting 4 d after initiation of partial feed restriction. Plasma NEFA was decreased for the highest dose: from 448 µEq/L to 138±75 µEq/L at 1 h after the first bolus of 3mg of NA/h per kilogram of BW. This initial reduction in plasma NEFA concentration was followed by an increase in concentration at 2, 3, and 4 h relative to initiation of infusions. Plasma NEFA then decreased to 243 µEq/L 6h after initiation of treatments and remained low until termination of infusions. A rebound in plasma NEFA concentration occurred at 3 and 4 h after termination of infusion for cows that received 3 mg of NA/h per kilogram of BW. Experiment 2 was a 5×5 Latin square with 7-d periods and 9 recovery days between each period. Each period consisted of 5 d of partial feed restriction to increase plasma NEFA concentration. Treatments were continuous abomasal infusion of 0, 0.5, 1, or 3 mg of free NA/h per kilogram of BW for 4.5 d starting at feed restriction or 0.5 mg of NA/h per kilogram of BW infused directly into the rumen in a form protected from microbial degradation. The ruminal administration of protected NA was initiated 2 d before abomasal infusions and initiation of feed restriction to establish steady postruminal delivery of NA by start of abomasal infusions. Plasma NEFA was approximately 70 µEq/L before initiation of feed restriction and increased to 509, 587, 442, 850, and 108 µEq/L at 4.5 d for cows that received 0, 0.5 (protected NA), 0.5 (free NA), 1, and 3 mg of NA/h per kilogram of BW, respectively. An antilipolytic response was achieved with the highest abomasal dose, which maintained plasma NEFA concentration lower than the control group. An increase in plasma NEFA concentration was observed after termination of infusions for cows that received 1 and 3 mg of NA/h per kilogram of BW. Plasma NEFA was 1,900 µEq/L at 4h after termination of infusion for cows receiving 1 mg of NA/h per kilogram of BW and 1,360 µEq/L at 5h after termination of infusion for cows receiving 3 mg of NA/h per kilogram of BW. In nongestating, nonlactating cows it is unlikely that a dose of NA exists that will reduce plasma NEFA concentration and prevent the rebound that occurs following termination of NA administration.

In vitro and in vivo analysis of fatty acid effects on metabolism of 17beta-estradiol and progesterone in dairy cows.

Some studies have reported improved reproductive performance with dietary fat supplementation. This study examined effects of fatty acids with different lengths, or desaturation, or both, on metabolism of estradiol (E2) and progesterone (P4) in bovine liver slice incubations (experiments 1 and 2) and in vivo (experiment 3). In experiment 1, effects of fatty acids C16:0 (palmitic acid), C16:1 (palmitoleic acid), C18:1 (oleic acid), and C18:3 (linolenic acid) were evaluated at 30, 100, and 300 microM on P4 and E2 metabolism in vitro. In experiment 2, stearic acid (C18:0) and C18:3 were evaluated in the same incubation conditions. In experiment 1, all of the fatty acids had some significant inhibitory effect on metabolism of P4, E2, or both (300 microM C16:0 on E2; 100 microM C16:1 on E2; 300 microM C16:1 on both P4 and E2; 300 microM C18:1 on P4; and 100 and 300 microM C18:3 on both P4 and E2). In experiment 2, C18:3 (100 and 300 microM) but not C18:0 decreased P4 and E2 metabolism. Overall, the most profound increase (approximately 60%) in half-life of P4 and E2 was observed with incubations of 300 microM C18:3 in both in vitro experiments. Based on these in vitro results, in experiment 3 linseed oil (rich in C18:3) was supplemented into the abomasum and acute effects on metabolism of E2 and P4 were evaluated. Cows (n=4) had endogenous E2 and P4 minimized (corpus luteum regressed, follicles aspirated) before receiving continuous intravenous infusion of E2 and P4 to analyze metabolic clearance rate for these hormones during abomasal infusion of saline (control) or 70 mL of linseed oil every 4h for 28h. Linseed oil infusion increased C18:3 in plasma by 46%; however, metabolic clearance rate for E2 and P4 were similar for control cows compared with linseed-treated cows. Thus, in vitro experiments indicated that E2 and P4 metabolism can be inhibited by high concentrations of C18:3. Nevertheless, in vivo, linseed oil did not acutely inhibit E2 and P4 metabolism, perhaps because insufficient C18:3 concentrations (increased to approximately 8 microM) were achieved. Further research is needed to determine the mechanism(s) of fatty acid inhibition of P4 and E2 metabolism and to discover practical methods to mimic this effect in vivo.

Effects of twin pregnancy and dry period feeding strategy on milk production, energy balance, and metabolic profiles in dairy cows.

The present study evaluated the interaction of pregnancy type [PT; single (S) vs. twin (T)] and dry period feeding management [D; close-up (CU) diet (NE(l) = 1.54 Mcal/kg of DM)] throughout the entire dry period (8W) vs. far-off (FO) diet (NE(l) = 1.32 Mcal/kg of DM) from 60 to 21 d before expected calving date (ECD) followed by CU diet until calving (3W). Treatments were arranged in a 2 x 2 factorial with a randomized block design with primiparous (n = 8) and multiparous (n = 39) Holstein cows. We hypothesized that increasing the duration of feeding a CU diet would improve metabolic status and lactation performance for cows with T, but not for cows with S. All cows were fed similarly in late lactation (90 to 60 d before ECD; diet NE(l) = 1.58 Mcal/kg of DM) and in early lactation (calving to 105 DIM; diet NE(l) = 1.71 Mcal/kg of DM). Prepartum DMI as percentage of BW did not differ (P > 0.10) with D but tended to be greater (P = 0.10) for cows with S than with T. Cows with T tended to have greater (P = 0.08) BW than cows with S, but conceptus-free BW was less (P = 0.001) for cows with T than for cows with S. No differences (P > 0.10) were detected in prepartum BCS or BCS change with PT or D. Energy balance (EB) was greater for cows with S than with T (P < 0.001) and for cows fed 8W vs. 3W (P = 0.01). Cows with T had greater (P < 0.001) NEFA and a tendency for greater liver triglycerides (TG; P = 0.07) and plasma beta-hydroxybutyrate (BHBA; P = 0.06) than cows with S. Prepartum cows fed 3W had greater (P = 0.01) liver TG and greater (P = 0.02) plasma NEFA, but less (P = 0.02) plasma BHBA than cows fed 8W. Plasma glucose (P < 0.004) and liver glycogen (P = 0.02) were less for cows with T but were not affected (P > 0.10) by D. Postpartum, there was no effect (P > 0.1) of PT or D on mean DMI as percentage of BW, BW, and BCS, but there was an interaction (P = 0.02) of PT x D for mean BCS. Cows that calved T were in a more positive (P = 0.004) EB than cows that calved S. Milk production was 5.2 kg/d greater (P = 0.04) for cows fed 8W; however, they were in less (P = 0.01) EB than cows that received 3W. Postpartum cows that calved T had decreased concentrations of plasma NEFA (P = 0.02) and liver TG (P = 0.04) but greater concentrations of plasma glucose (P = 0.03) than cows that calved S. Plasma BHBA (P = 0.07) and NEFA tended (P = 0.06) to be greater for cows that received 8W than 3W. Neither PT nor D affected (P > 0.1) plasma glucose and liver glycogen. There was a tendency for an interaction of PT x D for plasma NEFA and liver TG. In contrast to our hypothesis, response to D was independent of PT. Based on milk production data from the present experiment, 8W is a more desirable feeding strategy than 3W.

Effects of abomasal lipid infusion on liver triglyceride accumulation and adipose lipolysis during fatty liver induction in dairy cows.

The objective was to determine the effects of abomasal infusion of linseed oil on liver triglyceride (TG) accumulation and adipose tissue lipolysis during an experimental protocol for induction of fatty liver. Eight nonpregnant, nonlactating Holstein cows were randomly assigned to treatments in a replicated 4 x 4 Latin square design. Treatments were abomasal infusion of water (W), tallow (T), linseed oil (LO), or half linseed oil and half tallow (LOT) at a rate of 0.56 g/kg of body weight per day. Each experimental period consisted of a 4-d fast concurrent with administration of treatments into the abomasum in 6 equal doses per day (every 4 h). Cows were fed ad libitum for 24 d between periods of fasting and lipid infusion. Infusion of linseed oil (LO and LOT) increased alpha-linolenic acid (C18:3n-3) content in serum (12.2, 10.4, 4.2, and 4.6 g/100 g of fatty acids for LO, LOT, T, and W, respectively), but not in the nonesterified fatty acid (NEFA) fraction of plasma. Treatments had no effect on plasma NEFA concentrations. Abomasal infusion of lipid increased in vitro stimulated lipolysis in subcutaneous adipose tissue, compared with W (4,294, 3,809, 4,231, and 3,293 nmol of glycerol released x g(-1) tissue x 2 h(-1) for LO, LOT, T, and W, respectively), but there was no difference between fat sources. Hepatic TG accumulation over 4-d fast was 2.52, 2.60, 2.64, and 2.09 +/- 0.75 microg of TG/microg of DNA for W, LO, LOT, and T, respectively, which did not differ. Abomasal infusion of LO did not reduce liver TG accumulation, plasma NEFA concentration, or alter in vitro adipose tissue lipolysis when compared with T. These results contrast with a previous study involving i.v. infusion of lipid emulsion derived from LO. Discrepancies might be explained by the use of different administration routes and a relatively modest induction of liver TG accumulation in the current experiment.

Connexin expression pattern in the endometrium of baboons is influenced by hormonal changes and the presence of endometriotic lesions.

Experimentally induced endometriosis in baboons serves as an elegant model to discriminate between endometrial genes which are primarily associated with normal endometrial function and those that are changed by the presence of endometriotic lesions. Since connexin genes are characteristic of the hormonally regulated differentiation of the endometrium, we have examined connexin expression in baboon endometrium to delineate if they are altered in response to the presence of endometriotic lesions. Connexin expression in the endometrium of cycling baboons is similar to that of the human endometrium with Connexin(Cx)43 being primarily seen in the stromal compartment and Cx26 and Cx32 being present predominantly in the epithelium. Although Cx32 is up-regulated during the secretory phase, Cx26 and Cx43 are down-regulated. In the baboon model of induced endometriosis a change in connexin pattern was evident in the presence of endometriotic lesions. In the secretory phase, Cx26 and Cx32 are no longer present in the epithelium but Cx26 is now observed primarily in the stromal cells. Infusion of chorionic gonadotrophin in a manner that mimics blastocyst transit in utero failed to rescue the aberrant stromal expression of Cx26 that is associated with the presence of endometriotic lesions suggesting an impairment of the implantation process. The altered connexin pattern coupled with a loss of the channel protein in the epithelium and a gain of Cx26 in the stromal compartment suggests that the presence of lesions changes the uterine environment and thereby the differentiation programme. This aberrant expression of connexins may be an additional factor that contributes to endometriosis-associated infertility.

Effect of dry period length on reproduction during the subsequent lactation.

Days dry may influence reproductive measures such as days to first postpartum ovulation, days open, and pregnancy per artificial insemination (AI). Holstein cows (n = 781) from an approximately 3,000-cow commercial dairy operation were randomly assigned to 1 of 2 treatments with different targeted dry period (DP) lengths. Treatments were 1) a traditional DP of 55 d (T) or 2) a shortened DP of 34 d (S). All dry cows on T were fed a low-energy diet until 35 d before expected calving, and then at 34 d before expected calving, cows on T and S were fed a moderate energy diet until parturition. After parturition, all cows consumed the same diets that included a postcalving diet followed by a lactation diet. Actual days dry for each treatment were close to expected values, 34 and 56 d for S and T, respectively. Median days until first postpartum ovulation occurred sooner for S compared with T (35 vs. 43 d). The percentage of cows that were classified anovular by 70 d in milk (DIM) was more than 2-fold greater for cows on T than S (18 vs. 8%). Cows received AI after standing estrus starting at d 45, and the percentage of cows pregnant at 70 DIM tended to be greater for S than T; younger cows were similar (20.2 vs. 18.8%), but there was a difference between S and T in older cows (20.3 vs. 10.6%). Similarly, median days open tended to be fewer for cows on S than T. At 300 DIM, 85% of cows in both treatments were pregnant. Combining data from first and second service, pregnancies per AI were greater in older cows on S than T (32 vs. 24%). Thus, shortening the DP appeared to increase reproductive efficiency in older cows by shortening time to first ovulation, reducing numbers of anovular cows, and improving fertility. Future studies at more locations with varying reproductive management strategies are needed to confirm and provide the mechanistic basis for these results.

Effects of dry period length on milk production and health of dairy cattle.

Holstein cows (n = 781) in a commercial dairy herd were used in a randomized design to evaluate 2 dry period (DP) management strategies on milk production, milk components, milk quality, colostrum quality, and incidence of metabolic disorders. Cows were randomly assigned to a traditional 55 d (T) or shortened 34 d (S) DP. Cows assigned to T were fed a low-energy diet until 34 d before expected calving at which time all cows were fed a moderate-energy transition diet until calving. Postpartum, cows assigned to T produced more milk and tended to produce more solids-corrected milk than cows on S. Treatment differences in milk and solids-corrected milk yield were accounted for by cows in their second lactation. Milk fat percentage did not differ between treatments, but milk protein percentage was greater for cows assigned to S. Colostrum quality measured as IgG concentration did not differ between management strategies. Somatic cell score and cases of mastitis were not affected by management strategy. There was a tendency for prepartum nonesterified fatty acid (NEFA) to be lower for cows assigned to T compared with S. However, postpartum, cows assigned to S had significantly lower NEFA concentrations than those assigned to T. The incidences of ketosis, retained placenta, displaced abomasum, and metritis did not differ between treatments. Postpartum energy balance, as indicated by plasma NEFA, may have been improved for cows assigned to S; there was no detectable effect on animal health.

Effects of abomasal infusion of linseed oil on responses to glucose and insulin in holstein cows.

The objective was to study the effects of abomasal infusion of linseed oil, a source rich in n-3 C18:3, on whole-body response to insulin (experiment 1) and on insulin antilipolytic effects during feed restriction (experiment 2). In experiment 1, eight nonlactating, non-gestating cows were assigned to a crossover design, fed to meet maintenance requirements, and infused abomasally with either linseed oil (LIN) or tallow (TAL) at a rate of 0.54 g/kg of body weight per d for 5.5 d. Infusions were performed every 8 h during the first 3 d of each period and every 4 h thereafter. Intravenous glucose tolerance tests (IVGTT) were performed on d 5 of each period, followed by i.v. insulin challenges (IC) 12 h later. In experiment 2, six nonlactating, nongestating cows were assigned to a replicated 3 x 3 Latin square design. The experimental protocol included a water (WTR) treatment and feeding was suspended on d 3, leading to 50 and 62 h of feed restriction before IVGTT and IC, respectively. Clearance of glucose during IVGTT and IC was not affected by treatments in either experiment. However, LIN had an insulin sensitizing effect in experiment 1, because a lower insulin concentration led to the same clearance of glucose as TAL. In experiment 1, plasma nonesterified fatty acid (NEFA) concentration was low, reflecting a postprandial state, but NEFA was greater for LIN than TAL during IVGTT (108 vs. 88 +/- 4 microEq/L) and IC (133 vs. 83 +/- 9 microEq/L). In experiment 2, insulin concentrations during IVGTT did not differ across treatments. Basal plasma NEFA concentration before IVGTT tended to be greater for LIN than for TAL (612 vs. 508 microEq/L). Plasma NEFA clearance rate during IVGTT was greater for LIN than for TAL (2.8 vs. 2.5%/min), leading to a shorter time to reach half NEFA concentration (25 vs. 29 min) and greater absolute value of NEFA response area under the curve [AUC; -64,150 vs. -46,402 (microEq/L) x 180 min]. Data suggest that LIN enhanced the antilipolytic effects of insulin. Yet, other factors could have been involved because plasma NEFA concentration before IVGTT was 104 muEq/L greater for LIN than TAL for unknown reasons.

Reduction of plasma NEFA concentration by nicotinic acid enhances the response to insulin in feed-restricted Holstein cows.

The objective was to investigate the relationship between elevated plasma nonesterified fatty acid (NEFA) concentration and insulin resistance in Holstein cows. Six nonlactating, nongestating, ruminally cannulated Holstein cows were blocked by body condition score and randomly assigned to a sequence of 2 treatments in a crossover design. Cows were offered legume and grass hay ad libitum supplemented with minerals and vitamins and were allowed free access to water and a trace mineralized salt block. Mobilization of body reserves was stimulated by withdrawing forage for 48 h before initiation of treatments. Treatments consisted of 11 hourly abomasal infusions of water (control) or nicotinic acid (NA; 6 mg/h per kg of body weight) as an antilipolytic agent. Infusions of NA decreased plasma NEFA concentration from 545 microEq/L to approximately 100 microEq/L within 2 h after initiation of treatments, and differences were maintained throughout infusions. Intravenous glucose tolerance test was performed 8 h after initiation of treatments and was followed by 3 h of blood sampling. The reduction of plasma NEFA concentration led to significantly greater glucose clearance rate (1.9 vs. 1.2%/min) and to decreased glucose half-life (37 vs. 58 min), time to reach basal concentration (81 vs. 114 min) and glucose response area under the curve during 180 min of sampling [6,942 vs. 10,085 (microIU/mL) x 180 min]. Enhanced glucose clearance was achieved when plasma NEFA was reduced by NA, despite lower insulin concentration (70.0 vs. 97.9 +/- 13.4 microIU/mL) and a tendency for smaller insulin response area under the curve during 180 min of sampling [7,646 vs. 12,104 +/- 2,587 (microIU/mL) x 180 min], reflecting an increased response to endogenous insulin. Based on literature, we do not expect NA to have altered glucose metabolism directly; therefore, this experiment demonstrates a cause and effect relationship between elevated NEFA and insulin resistance in Holstein cows.

The use of nicotinic acid to induce sustained low plasma nonesterified fatty acids in feed-restricted Holstein cows.

The objectives were to determine the effects of nicotinic acid (NA) on blood metabolites (experiment 1) and whether successive doses of NA could induce sustained reductions of plasma nonesterified fatty acids (NEFA; experiment 2) in feed-restricted, nonlactating Holstein cows. Experiment 1 was a single 4 x 4 Latin square with 1-wk periods. Each period consisted of 2.5 d of feed restriction to increase plasma NEFA and 4.5 d of ad libitum feeding. Treatments were abomasal administration of 0, 6, 30, or 60 mg of NA/kg of body weight (BW), given as a single bolus 48 h after initiation of feed restriction. Plasma NEFA concentration decreased from 546 microEq/L to 208 +/- 141 microEq/L at 1 h after the infusion of 6 mg of NA/kg of BW, and to less than 100 +/- 148 microEq/L at 3 h after the abomasal infusion of the 2 highest doses of NA. A rebound occurred after the initial decrease of plasma NEFA concentration. The rebound lasted up to 9 h for the 30-mg dose of NA, and up to 6 h for the 6-mg dose. Experiment 2 was a randomized complete block design with 3 treatments and 6 cows. Starting at 48 h of feed restriction, cows received 9 hourly abomasal infusions of 0, 6, or 10 mg of NA/kg of BW. Plasma NEFA concentrations decreased from 553 microEq/L +/- 24 immediately before the initiation of treatments to <100 microEq/L during hourly infusions of 6 or 10 mg of NA/kg. Data suggest that the maximal antilipolytic response was achieved with the lowest dose of NA. A rebound of NEFA started 2 to 3 h after NA infusions were terminated. In both experiments, the NEFA rebound period coincided with increases in insulin and no change or increased glucose concentrations, suggesting a state of insulin resistance induced by elevated NEFA. This model for reducing plasma NEFA concentration by abomasal infusions of NA can be used to study the metabolic ramifications of elevated vs. reduced NEFA concentrations. The data demonstrate potential benefits and pitfalls of using NA to regulate plasma NEFA and prevent lipid-related metabolic disorders.

Induction of hyperlipidemia by intravenous infusion of tallow emulsion causes insulin resistance in Holstein cows.

The objective was to test whether the induction of elevated blood nonesterified fatty acids (NEFA) by i.v. infusion of a tallow emulsion altered glucose tolerance and responsiveness to insulin in Holstein cows. Six non-lactating, nongestating Holstein cows were assigned to a crossover design. One cow was excluded before initiation of the experiment because of complications from mastitis. Treatments consisted of 11-h i.v. infusions of saline (control) or a 20% (wt/vol) triacylglycerol (TG) emulsion derived from tallow (tallow) to elevate plasma NEFA. Each period consisted of two 11-h infusions (INF1 and INF2), separated by 1 d in which cows were not infused. Intravenous glucose tolerance tests (IVGTT) and insulin challenges (IC) were performed 8 h after initiation of INF1 and INF2, respectively. The infusion of treatments continued during the 3 h of sampling for IVGTT and IC. Cows were fed every 4 h at a rate to meet energy requirements for 5 d prior to each period, and every 2 h during the first 8 h of infusions. Infusion of tallow induced hyperlipidemia by increasing plasma NEFA (295 +/- 9 vs. 79 +/- 7 microEq/L), serum TG (41.0 +/- 6 vs. 11.4 +/- 4.4 mg/dL), and glycerol (0.81 +/- 0.09 vs. 0.23 +/- 0.1 mg/dL) concentrations during INF1. During INF2, tallow treatment increased plasma NEFA (347 vs. 139 +/- 18 microEq/L), serum TG (20.8 +/- 4.6 vs. 13.1 +/- 2.3 mg/dL), and glycerol (0.88 +/- 0.04 vs. 0.31 +/- 0.02 mg/dL) concentrations. Induction of hyperlipidemia impaired glucose clearance during IVGTT, despite the greater endogenous insulin response to the glucose infusion, leading to a lower insulin sensitivity index [0.29 vs. 1.88 +/- 0.31 x 10(-4) min(-1)/(microIU/mL)]. Accordingly, hyperlipidemia impaired glucose clearance during IC (1.58 vs. 2.72 %/min), reflecting lower responsiveness to insulin. These data show that induction of hyperlipidemia causes insulin resistance in Holstein cows by impairing both sensitivity and maximum responsiveness to insulin. The induction of insulin resistance by TG, NEFA, or both may increase the availability of glucogenic nutrients to the periparturient dairy cow. Yet excessive elevation of NEFA may potentially lead adipocytes to become more insulin resistant, further increasing plasma NEFA concentration and the risk of metabolic disorders.

Strategies to improve fertility of high yielding dairy farms: Management of the dry period.

Reproductive performance of dairy cattle has been related to a wide variety of indicators of energy status, e.g., extent of negative energy balance, time of energy balance nadir, body weight loss, body condition score, and body condition score loss. Energy balance begins to decrease during the last few weeks prior to calving primarily due to a 30-35% reduction in feed intake. Cows typically remain in negative energy balance for five to seven weeks postpartum. Nutritional strategies to improve energy balance during the transition period include fat supplementation and feeding additional nonfiber carbohydrate. Unfortunately, neither approach is likely to markedly enhance energy status, although fat supplementation may increase reproductive efficiency independent of any effect on energy balance. Alternative management strategies may be required to improve fertility of dairy cows. Shortening or eliminating the dry period may improve energy status of dairy cows and increase reproductive efficiency. Shortening or eliminating the dry period may enhance dry matter intake during the transition period, decrease milk energy output, or both. A preliminary study using small animal numbers indicated that reducing dry period length to 28 or 0 days may decreases days to first ovulation, increase first service conception rate, and decrease days open. A follow-up study employing large animal numbers confirmed that reducing dry period length from 55 to 34 days can decrease days to first ovulation and decrease the percentage of anovular cows. The reduction in days open was greater for older cows than second parity cows. The reduction in days open was not related to effects of treatment on milk yield. Shortening or eliminating the dry period may be a more successful approach to improving reproductive efficiency than diet manipulation.

Technical note: Effect of sampling protocol on plasma nonesterified fatty acid concentration in dairy cows.

The objective of these experiments was to determine effects of sampling protocol on plasma nonesterified fatty acid (NEFA) concentration. In experiment 1, 8 nonlactating, nongestating dairy cows were blood sampled from a jugular vein catheter (basal, 0 min), moved to an exercise lot for 15 min, returned to stanchions, and sampled immediately and at 5, 15, 30, 60, and 120 min following return to their stalls. Following 15 min of exercise, plasma NEFA concentration increased, peaking at 5 min (225 microEq/L) and returning to basal (84 microEq/L) by 30 min (110 microEq/L). Cows were then moved to box stalls overnight, and 24 h after the basal sample, they were locked up and sampled again. Housing cows in a box stall overnight and locking them in headlocks increased plasma NEFA concentration (184 microEq/L). In a second experiment at a large free-stall commercial dairy, 11 late-gestation nonlactating dairy cows were locked in headlocks at feeding, blood was sampled from the coccygeal artery or vein (0 min), and cows were then released and allowed to finish eating and return to their stalls. Cows were then herded to headlocks and sampled immediately at 120 min after initial sampling and at 135, 150, and 180 min. Plasma NEFA concentration was highest at initial lockup (0 min; 284 microEq/L), lowest at 180 min (178 microEq/L), and intermediate at time points in between. A second group of 10 late-gestation nonlactating dairy cows were locked in headlocks at feeding, and blood was sampled immediately and at 5, 15, 30, and 60 min. Plasma NEFA concentration was highest 15 min after being placed in headlocks and lowest 60 min after lockup (221 and 113 microEq/L, respectively). At each time point in experiments 1 and 2, a behavior score was given (1 to 10; 1 = calm; 10 = extremely excited). In both experiments, there was a significant correlation between the plasma NEFA concentration and behavior score. In conclusion, plasma NEFA concentration was affected by sampling protocol.