Duration and degree of diet-induced metabolic acidosis prepartum alter tissue responses to insulin in dairy cows.

The objective of this experiment was to determine the effects of altering the dietary cation-anion difference (DCAD) fed for the last 21 or 42 d of gestation on glucose metabolism and tissue insulin responsiveness. Ninety parous Holstein cows at 232 d of gestation were assigned randomly to dietary treatments with 2 levels of DCAD (-70 or -180 mEq/kg) fed for 2 durations (short: the last 21 d of gestation; long: the last 42 d of gestation). For the short treatments, a diet with +110 mEq/kg was fed from 232 to 254 d of gestation. Intravenous glucose tolerance tests (IVGTT) were performed at either 250 or 270 d of gestation by infusing 0.25 g of dextrose/kg of body weight within 1 min. The following day, cows underwent an insulin challenge (IC) and received 0.1 IU of insulin/kg of body weight intravenously. Blood was sampled at min -15, -5, and 0 to establish a baseline and from 5 to 180 min relative to infusions; plasma concentrations of glucose, insulin, and fatty acids were determined, and the respective areas under the curves (AUC) were calculated. Liver was sampled after the IVGTT, and adipose tissue was sampled after the IVGTT and IC for quantification of mRNA expression and protein abundance. Reducing the DCAD altered acid-base balance compatible with a compensated metabolic acidosis. At 250 d, reducing the DCAD increased the AUC for glucose and reduced that of insulin following the IVGTT, whereas during the IC, clearance rate decreased and time to half-life of insulin increased with reducing DCAD, resulting in a tendency to a larger AUC for fatty acids. At 270 d, quantitative insulin sensitivity check index and the revised quantitative insulin sensitivity check index were smaller in cows fed the acidogenic diets for the last 42 d of gestation compared with the last 21 d of gestation, thereby suggesting reduced insulin sensitivity. In addition, cows fed for the long duration tended to have greater AUC for glucose but smaller AUC for insulin following an IVGTT than those fed for the short duration, thereby suggesting reduced insulin release and glucose disposal. Treatments did not affect hepatic mRNA expression of G6PC, PCK1, PCK2, and PC or adipose tissue mRNA expression of ATGL, ACC, B2AR, HSL, and PLIN1. On the other hand, for proteins, reducing the DCAD linearly reduced abundance of rabbit anti-mouse protein kinase B (AKT) and tended to reduce rabbit anti-human phosphorylated (Ser-9) glycogen synthase kinase-3 ß (pGSK) and the pGSK:rabbit anti-human glycogen synthase kinase-3 ß (GSK) ratio in hepatic tissue, whereas a linear increase in rabbit anti-human hormone-sensitive lipase (HSL) and rabbit anti-mouse phosphorylated (Ser-660) hormone-sensitive lipase (pHSL) in adipose tissue was observed after the IVGTT at 250 d. Moreover, reducing the DCAD resulted in a linear reduction of AKT and tended to reduce rabbit anti-human acetyl-CoA carboxylase (ACC) but increased pHSL linearly in adipose tissue after an IC at 250 d. Cows fed acidogenic diets for a short duration tended to have less pHSL in adipose tissue than those fed for a long duration after an IVGTT at 270 d. Associations were observed between blood pH and mRNA and protein abundance in hepatic and adipose tissues. Diet-induced metabolic acidosis altered insulin release and insulin signaling, resulting in a shift in adipose tissue metabolism that would favor lipolysis over lipogenesis.

Increasing the length of an estradiol with progesterone timed artificial insemination protocol with 2 controlled internal drug release devices improves pregnancy per artificial insemination in lactating dairy cows.

The objective of this study was to compare the effects of different lengths of ovulation synchronization protocols using 2 controlled internal drug release (CIDR) devices on ovarian dynamics and pregnancy outcomes in lactating dairy cows. Lactating Holstein cows (n = 1,979) were randomly assigned to receive timed artificial insemination (TAI; d 0) following 1 of 2 treatments: (1) 9-d protocol (n = 988; 9D) with 2 intravaginal devices containing 1.9 g of progesterone (CIDR) and 2.0 mg of estradiol benzoate on day -11; 25 mg (i.m.) of dinoprost tromethamine (PG) and withdrawal of 1 CIDR on d -4; 1.0 mg (i.m.) of estradiol cypionate, second CIDR withdrawal, and PG on d -2; and TAI on d 0 and (2) 10-d protocol (n = 991; 10D) with 2 CIDR and 2.0 mg of estradiol benzoate on d -12; 25 mg of PG and withdrawal of 1 CIDR on d -4; 1.0 mg of estradiol, second CIDR withdrawal, and PG on d -2; and TAI on d 0. There was no effect of protocol on estrus detection, whereas a greater percentage of cows from 10D had ovulated close to TAI [no corpus luteum (CL) at AI and a CL at d 7] versus cows assigned to 9D protocol. A protocol × heat stress (average cow temperature ≥39.1°C on day of AI and d 7) interaction was observed in a manner that pregnancy per AI (P/AI) was greater in non-heat-stressed 10D versus 9D cows, whereas P/AI did not differ when cows were under heat stress. Furthermore, 10D protocol did not increase P/AI when all cows that received AI were included in the analysis or in cows that ovulated near TAI. However, animals assigned to 9D without any event of heat stress had a reduced P/AI when compared with cows assigned to 10D without heat stress. A protocol × CL presence at the beginning of the protocol interaction was observed and cows with a CL at the beginning of the protocol had a greater P/AI in 10D versus 9D; meanwhile, in cows without a CL, no differences on P/AI were observed. The protocol × CL presence at the beginning of the protocol interaction on P/AI was also observed for cows that ovulated near TAI. A greater percentage of cows assigned to 9D had follicles of medium size (13-15.9 mm), and greater percentage of cows assigned to 10D had larger follicles (>16 mm). Increasing the length of an estradiol with progesterone-based ovulation synchronization protocol (10D vs. 9D) increased the proportion of cows with larger follicles (>16 mm) and increased P/AI in cows without heat stress and in cows with a CL at beginning of the protocol. Moreover, the 10D protocol increased the proportion of cows with ovulation near TAI, demonstrating the effectiveness of this protocol in improving the reproductive performance of lactating Holstein cows.

Prostaglandin F2α influences pre-ovulatory follicle characteristics and pregnancy per AI in anovular dairy cows.

Objectives were to determine the effects of a dose of PGF2α administered 2 days before timed artificial insemination (AI) on LH pulsatility, characteristics of the pre-ovulatory follicle, and pregnancy per artificial insemination (P/AI) in anovular dairy cows, particularly in cows not subjected to hyperthermia. In experiment 1, 2,011 lactating Holstein cows had ovaries scanned by ultrasound to determine corpus luteum (CL) presence and only those without a CL in two consecutive exams were enrolled (n = 437). Cows had the estrous cycle synchronized with an estradiol-progesterone based protocol starting on experiment Day -11 and timed AI on Day 0. Cows were assigned randomly to receive a single dose of 25 mg of PGF2α as dinoprost on Day -4 (1PGF, n = 222) or two doses of 25 mg each of PGF2α, one on Day -4 and one on Day -2 (2PGF, n = 215). Rectal temperatures were evaluated on the day of AI and 7 days later and cows were classified as being normothermic (<39.1 °C) or hyperthermic (≥39.1 °C). Ovulatory responses and P/AI were determined. In experiment 2, cows with regressed CL were exposed to low concentrations of progesterone and then randomly assigned to the same estrous synchronization protocol and treatments, 1PGF (n = 28) and 2PGF (n = 28). Blood was sampled and analyzed for concentrations of progesterone, and for concentrations of LH and 13,14-dihydro-15-keto-PGF2α metabolite (PGFM) every 15 min starting 1 h before to 6 h after treatments and then every 2 h from 12 to 59 h after treatments. The pre-ovulatory follicle was aspirated 44 h after treatments and concentrations of estradiol quantified. In experiment 1, treatment of anovular cows with a second dose of PGF2α increased P/AI in normothermic cows (19.8 [18/91] vs. 38.8% [31/80]), but not in hyperthermic cows. Synchronization was not affected by treatment, but it was greater for normothermic than hyperthermic cows (87.1 [149/171] vs. 77.8% [207/266]). When only synchronized cows were evaluated, the same responses were observed; treatment with 2PGF increased P/AI compared with 1PGF in normothermic cows (23.1 [18/78] vs. 43.7% [31/71]), but not in hyperthermic cows. In experiment 2, administration of 25 mg of dinoprost in 2PGF resulted in concentrations of PGFM 26-fold greater than 1PGF in the first 6 h after treatment (48 vs. 1,242 pg/mL). Cows receiving 2PGF had smaller basal LH concentration (0.57 vs. 0.46 ng/mL) and less frequent LH pulses (4.5 vs. 3.9 pulses/6 h), but duration of the LH surge was longer for 2PGF than 1PGF (13.1 vs. 15.5 h). Treatment with 2PGF increased the diameter and volume of the pre-ovulatory follicle, and concentration of estradiol (115 vs. 262 ng/mL) and total follicular estradiol content (124 vs. 505 ng) compared with 1PGF. Collectively, these results suggest that PGF2α has a role in fertility of anovular cows that is unrelated to its luteolytic effect.

Evaluation of presynchronization and addition of GnRH at the beginning of an estradiol/progesterone protocol on circulating progesterone and fertility of lactating dairy cows.

The objectives of this study were to determine if the utilization of a presynchronization strategy would improve fertility at first artificial insemination (AI) during an E2/P4 ovulation synchronization protocol with or without GnRH administration at the beginning of the protocol. This experiment was conducted using cows (n = 665) at their first postpartum service and the following breeding treatment: CIDR insertion and 2.0 mg of estradiol benzoate (EB) on day -11; 25 mg dinoprost tromethamine (PG) on day -4; PG, CIDR withdrawal, and 1.0 mg of estradiol cypionate (ECP) on day -2; timed-AI on day 0. At 31 ± 3 days postpartum, cows were randomly allocated to one of three treatments on a weekly basis: 1) -P + GnRH: cows assigned to the breeding protocol with 100 µg of GnRH on day -11, 2) P + GnRH: cows assigned to a presynchronization protocol using CIDR insertion +2 mg EB on day -28, PG + ECP and CIDR withdrawal on day -21, and beginning of the breeding protocol plus GnRH (100 µg) on day -11, and 3) +P-GnRH: cows assigned to a presynchronization protocol and the breeding treatment without GnRH on day -11. No treatment effects were observed on P/AI at the pregnancy diagnoses on days 32 and 60, or for pregnancy losses between days 32 and 60 of pregnancy whether analyses included all cows or only cows that ovulated near TAI. Moreover, milk yield negatively affected P/AI. Cows with greater circulating P4 concentrations on day -4 had greater P/AI on day 60. Cows without CL on day -11 had a reduced P/AI and this effect was more significant in cows not treated with GnRH. Cows assigned to -P + GnRH had the lowest circulating P4 concentration on day -4 (3.4 ± 0.16 ng/mL), followed by + P-GnRH (4.56 ± 0.17 ng/mL), and +P + GnRH (5.08 ± 0.17 ng/mL) cohorts. The data of the current study suggest that the combination of a Presynch and GnRH administration at the beginning of a TAI protocol was the most effective way to increase the % of cows with a functional CL and with elevated circulating P4 concentrations at the time of PG treatment.

Effects of level of dietary cation-anion difference and duration of prepartum feeding on performance and metabolism of dairy cows.

The objectives were to evaluate the effects of feeding diets with 2 levels of negative dietary cation-anion differences (DCAD) during the last 42 or 21 d of gestation on performance and metabolism in dairy cows. The hypothesis was that extending feeding from 21 to 42 d and reducing the DCAD from -70 to -180 mEq/kg of dry matter (DM) would not be detrimental to performance. Holstein cows at 230 d of gestation were blocked by parity prepartum (48 entering their second lactation and 66 entering their third or greater lactation) and 305-d milk yield, and randomly assigned to 1 of 4 treatments arranged as a 2 × 2 factorial. The 2 levels of DCAD, -70 or -180 mEq/kg of DM, and 2 feeding durations, the last 21 d (short) or the last 42 d (long) prepartum resulted in 4 treatments, short -70 (n = 29), short -180 (n = 29), long -70 (n = 28) and long -180 (n = 28). Cows in the short treatments were fed a diet with DCAD of +110 mEq/kg of DM from -42 to -22 d relative to calving. After calving, cows were fed the same diet and production and disease incidence were evaluated for 42 d in milk, whereas reproduction and survival was evaluated for 305 d in milk. Blood was sampled pre- and postpartum for quantification of metabolites and minerals. Reducing the DCAD linearly decreased prepartum DM intake between -42 and -22 d relative to calving (+110 mEq/kg of DM = 11.5 vs. -70 mEq/kg of DM = 10.7 vs. -180 mEq/kg of DM = 10.2 ± 0.4), and a more acidogenic diet in the last 21 d of the dry period reduced intake by 1.1 kg/d (-70 mEq/kg of DM = 10.8 vs. -180 mEq/kg of DM = 9.7 ± 0.5 kg/d). Cows fed the -180 mEq/kg of DM diet had increased concentrations of ionized Ca in blood on the day of calving (-70 mEq/kg of DM = 1.063 vs. -180 mEq/kg of DM = 1.128 ± 0.020 mM). Extending the duration of feeding the diets with negative DCAD from 21 to 42 d reduced gestation length by 2 d (short = 277.2 vs. long = 275.3 d), milk yield by 2.5 kg/d (short = 40.4 vs. long = 37.9 ± 1.0 kg/d) and tended to increase days open because of reduced pregnancy per artificial insemination (short = 35.0 vs. long = 22.6%). Results suggest that increasing the duration of feeding diets with negative DCAD from 21 to 42 d prepartum might influence milk yield and reproduction of cows in the subsequent lactation, although yields of 3.5% fat- and energy-corrected milk did not differ with treatments. Reducing the DCAD from -70 to -180 mEq/kg of DM induced a more severe metabolic acidosis, increased ionized Ca concentrations prepartum and on the day of calving, and decreased colostrum yield in the first milking, but had no effects on performance in the subsequent lactation. Collectively, these data suggest that extending the feeding of an acidogenic diet beyond 21 d is unnecessary and might be detrimental to dairy cows, and a reduction in the DCAD from -70 to -180 mEq/kg of DM is not needed.

Comparison of 2 protocols to increase circulating progesterone concentration before timed artificial insemination in lactating dairy cows with or without elevated body temperature.

Two treatments designed to increase circulating progesterone concentration (P4) during preovulatory follicle development were compared. One treatment used 2 intravaginal P4 implants (controlled internal drug-releasing inserts; CIDR) and the other used a GnRH treatment at beginning of the protocol. Lactating Holstein cows that had been diagnosed as nonpregnant were randomly assigned to receive timed artificial insemination (TAI) following 1 of 2 treatments (n = 1,638 breedings): (1) GnRH: CIDR+ 2 mg of estradiol (E2) benzoate + 100 µg of GnRH on d -11, PGF2α on d -4, CIDR withdrawal + 1.0 mg of E2-cypionate + PGF2α) on d -2, and TAI on d 0; or (2) 2CIDR: 2 CIDR + 2 mg of E2-benzoate on d -11, 1 CIDR withdrawn + PGF2α on d -4, second CIDR withdrawn + 1.0 mg of E2-cypionate + PGF2α on d -2, and TAI on d 0. Milk yield was measured daily between d 0 and d 7. Rectal temperature was measured using a digital thermometer at d 0 and 7, and elevated body temperature was defined as an average rectal temperature ≥39.1°C. Pregnancy diagnoses were performed on d 32 and 60 after TAI. We detected no effect of treatments on pregnancy per AI or pregnancy loss regardless of elevated body temperature, body condition score, parity, milk yield, or presence or absence of a corpus luteum (CL) on d -11 or d -4. Pregnancy per AI at 60 d was reduced [elevated body temperature = 22.8% (162/709), no elevated body temperature 34.1% (279/817)] and pregnancy loss tended to increase [elevated body temperature = 20.2% (41/203), no elevated body temperature 14.4% (47/326)] in cows with elevated body temperature. Various physiological measurements associated with greater fertility were also reduced in cows with elevated body temperature, such as percentage of cows with a CL at PGF2α (decreased 7.9%), ovulatory follicle diameter (decreased 0.51 mm), expression of estrus (decreased 5.1%), and ovulation near TAI (decreased 2.8%) compared with cows without elevated body temperature. A greater proportion of cows (30.2%) had a CL at PGF2α in the GnRH treatment [74.1% (570/763)] than in the 2CIDR treatment [56.9% (434/763)]; however, circulating P4 concentration was greater at the time of PGF2α treatment (d -4) for cows 2CIDR (4.26 ± 0.13 ng/mL) than in cows in GnRH (3.99 ± 0.14 ng/mL). Thus, these 2 protocols yield similar fertility results that might be due to somewhat different physiological alterations. Treatment with GnRH increased the proportion of cows with a CL at PGF2α; however, the 2CIDR protocol increased circulating P4 under all circumstances.

Circulating concentrations of bovine pregnancy-associated glycoproteins and late embryonic mortality in lactating dairy herds.

The objectives of these experiments were as follows: (1) to determine the association between circulating concentrations of pregnancy-associated glycoproteins (PAG) and late embryonic mortality (EM) in lactating dairy cattle following fixed-time artificial insemination (TAI) on d 0 or timed embryo transfer (TET) on d 7, (2) to identify a circulating concentration of PAG on d 31 below which late EM would be likely to occur, and (3) to identify when during gestation (d 31-59) late EM is occurring. Cows were diagnosed pregnant on d 31 of gestation based on presence of a fetal heartbeat and reconfirmed to be pregnant on d 59 of gestation. Late EM occurred when a cow had a viable embryo on d 31 of gestation but not on d 59 following TAI or TET. Only pregnant cows on d 31 were included in the analysis (TAI-maintained, n=413; TAI-EM, n=77; TET-maintained, n=238; TET-EM, n=47). Cows that were pregnant at d 31 of gestation and maintained the pregnancy until d 59 had significantly higher circulating concentrations of PAG at d 31 of gestation compared with cows that experienced late EM between d 31 and 59 of gestation in both TAI and TET. To conduct a more stringent test of the effectiveness of a single circulating PAG concentration (d 31) to predict EM, a receiver-operating characteristic curve was generated to identify a PAG concentration on d 31 that would predict EM with ≥95% accuracy in cows that received TAI or TET. Based on positive and negative predicative value analysis, a circulating concentration of PAG below 1.4 ng/mL (TAI; minimal detectable level 0.28 ng/mL) and 1.85 ng/mL (TET) was 95% accurate in predicting EM (between d 31 and 59) at d 31 of gestation, respectively. Following TET, embryonic loss was tracked by Doppler ultrasound, progesterone, and PAG from d 24 to 59 of gestation, with more than 50% of the loss occurring between d 31 and 38 of gestation. In summary, circulating concentrations of PAG on d 31 of gestation may provide a good marker for predicting EM between d 31 and 59 of gestation, and the data suggest that this model could help predict which cows will undergo late EM.

Double gene therapy with granulocyte colony-stimulating factor and vascular endothelial growth factor acts synergistically to improve nerve regeneration and functional outcome after sciatic nerve injury in mice.

Peripheral-nerve injuries are a common clinical problem and often result in long-term functional deficits. Reconstruction of peripheral-nerve defects is currently undertaken with nerve autografts. However, there is a limited availability of nerves that can be sacrificed and the functional recovery is never 100% satisfactory. We have previously shown that gene therapy with vascular endothelial growth factor (VEGF) significantly improved nerve regeneration, neuronal survival, and muscle activity. Our hypothesis is that granulocyte colony-stimulating factor (G-CSF) synergizes with VEGF to improve the functional outcome after sciatic nerve transection. The left sciatic nerves and the adjacent muscle groups of adult mice were exposed, and 50 or 100 µg (in 50 µl PBS) of VEGF and/or G-CSF genes was injected locally, just below the sciatic nerve, and transferred by electroporation. The sciatic nerves were transected and placed in an empty polycaprolactone (PCL) nerve guide, leaving a 3-mm gap to challenge nerve regeneration. After 6 weeks, the mice were perfused and the sciatic nerve, the dorsal root ganglion (DRG), the spinal cord and the gastrocnemius muscle were processed for light and transmission electron microscopy. Treated animals showed significant improvement in functional and histological analyses compared with the control group. However, the best results were obtained with the G-CSF+VEGF-treated animals: quantitative analysis of regenerated nerves showed a significant increase in the number of myelinated fibers and blood vessels, and the number of neurons in the DRG and motoneurons in the spinal cord was significantly higher. Motor function also showed that functional recovery occurred earlier in animals receiving G-CSF+VEGF-treatment. The gastrocnemius muscle showed an increase in weight and in the levels of creatine phosphokinase, suggesting an improvement of reinnervation and muscle activity. These results suggest that these two factors acted synergistically and optimized the nerve repair potential, improving regeneration after a transection lesion.

Enhancement of sciatic nerve regeneration after vascular endothelial growth factor (VEGF) gene therapy.

AIMS: Recent studies have emphasized the beneficial effects of the vascular endothelial growth factor (VEGF) on neurone survival and Schwann cell proliferation. VEGF is a potent angiogenic factor, and angiogenesis has long been recognized as an important and necessary step during tissue repair. Here, we investigated the effects of VEGF on sciatic nerve regeneration. METHODS: Using light and electron microscopy, we evaluated sciatic nerve regeneration after transection and VEGF gene therapy. We examined the survival of the neurones in the dorsal root ganglia and in lumbar 4 segment of spinal cord. We also evaluated the functional recovery using the sciatic functional index and gastrocnemius muscle weight. In addition, we evaluated the VEGF expression by immunohistochemistry. RESULTS: Fluorescein isothiocyanate-dextran (FITC-dextran) fluorescence of nerves and muscles revealed intense staining in the VEGF-treated group. Quantitative analysis showed that the numbers of myelinated fibres and blood vessels were significantly higher in VEGF-treated animals. VEGF also increased the survival of neurone cell bodies in dorsal root ganglia and in spinal cord. The sciatic functional index and gastrocnemius muscle weight reached significantly higher values in VEGF-treated animals. CONCLUSION: We demonstrate a positive relationship between increased vascularization and enhanced nerve regeneration, indicating that VEGF administration can support and enhance the growth of regenerating nerve fibres, probably through a combination of angiogenic, neurotrophic and neuroprotective effects.