Relationships between gastrointestinal permeability, heat stress, and milk production in lactating dairy cows.

Heat stress (HS) is a global issue that decreases farm profits and compromises animal welfare. To distinguish between the direct and indirect effects of HS, 16 multiparous Holstein cows approximately 100 DIM were assigned to one of 2 treatments: pair fed to match HS cow intake, housed in thermoneutral conditions (PFTN, n = 8) or cyclical HS (n = 8). All cows were subjected to 2 experimental periods. Period 1 consisted of a 4 d thermoneutral period with ad libitum intake. During period 2 (P2), the HS cows were housed in cyclical HS conditions with a temperature-humidity index (THI) ranging from 76 to 80 and the PFTN cows were exposed to a constant THI of 64 for 4 d. Dry matter intake of the PFTN cows was intake matched to the HS cows. Milk yield, milk composition, rectal temperature, and respiration rate were recorded twice daily, blood was collected daily via a jugular catheter, and cows were fed twice daily. On d 3 of each period, Cr-EDTA and sucralose were orally administered and recovered via 24 h total urine collection to assess gastrointestinal permeability. All data were analyzed using the GLIMMIX procedure in SAS. The daily data collected in P1 was averaged and used as a covariate if deemed significant in the model. Heat stress decreased voluntary feed intake by 35% and increased rectal temperature and respiration rate (38.4°C vs. 39.4°C and 40 vs. 71 respirations/min, respectively). Heat stress reduced DMI by 35%, which accounted for 66% of the decrease in milk yield. The yields, and not concentrations, of milk protein, fat, and other solids were lower in the HS cows on d 4 of P2. Milk urea nitrogen was higher and plasma urea nitrogen tended to be higher on d 3 and d 4 of HS. Glucose was 7% lower in the HS cows and insulin was 71% higher in the HS cows than the PFTN cows on d 4 of P2. No difference in lipopolysaccharide-binding protein was observed. Heat stress cows produced 7 L/d more urine than PFTN cows. No differences were detected in the urine concentration or percentage of the oral dose recovered for Cr-EDTA or sucralose. In conclusion, HS was responsible for 34% of the reduction of milk yield. The elevated MUN and the tendency for elevated plasma urea nitrogen indicate a whole-body shift in nitrogen metabolism. No differences in gastrointestinal permeability or lipopolysaccharide-binding protein were observed. These results indicate that, under the conditions of this experiment, activation of the immune system by gut-derived lipopolysaccharide was not responsible for the decreased milk yield observed during HS.

Intramammary lipopolysaccharide challenge in early- versus mid-lactation dairy cattle: Immune, production, and metabolic responses.

Study objectives were to compare the immune response, metabolism, and production following intramammary LPS (IMM LPS) administration in early and mid-lactation cows. Early (E-LPS; n = 11; 20 ± 4 DIM) and mid- (M-LPS; n = 10; 155 ± 40 DIM) lactation cows were enrolled in an experiment consisting of 2 periods (P). During P1 (5 d) cows were fed ad libitum and baseline data were collected, including liver and muscle biopsies. At the beginning of P2 (3 d) cows received 10 mL of sterile saline containing 10 µg of LPS from Escherichia coli O111:B4/mL into the left rear quarter of the mammary gland, and liver and muscle biopsies were collected at 12 h after LPS. Tissues were analyzed for metabolic flexibility, which measures substrate switching capacity from pyruvic acid to palmitic acid oxidation. Data were analyzed with the MIXED procedure in SAS 9.4. Rectal temperature was assessed hourly for the first 12 h after LPS and every 6 h thereafter for the remainder of P2. All cows developed a febrile response following LPS, but E-LPS had a more intense fever than M-LPS cows (0.7°C at 5 h after LPS). Blood samples were collected at 0, 3, 6, 9, 12, 24, 36, 48, and 72 h after LPS for analysis of systemic inflammation and metabolism parameters. Total serum Ca decreased after LPS (26% at 6 h nadir) but did not differ by lactation stage (LS). Circulating neutrophils decreased, then increased after LPS in both LS, but E-LPS had exaggerated neutrophilia (56% from 12 to 48 h) compared with M-LPS. Haptoglobin increased after LPS (15-fold) but did not differ by LS. Many circulating cytokines were increased after LPS, and IL-6, IL-10, TNF-α, MCP-1, and IP-10 were further augmented in E-LPS compared with M-LPS cows. Relative to P1, all cows had reduced milk yield (26%) and DMI (14%) on d 1 that did not differ by LS. Somatic cell score increased rapidly in response to LPS regardless of LS and gradually decreased from 18 h onwards. Milk component yields decreased after LPS. However, E-LPS had increased fat (11%) and tended to have increased lactose (8%) yield compared with M-LPS cows throughout P2. Circulating glucose was not affected by LPS. Nonesterified fatty acids (NEFA) decreased in E-LPS (29%) but not M-LPS cows. ß-Hydroxybutyrate slightly increased (14%) over time after LPS regardless of LS. Insulin increased after LPS in all cows, but E-LPS had blunted hyperinsulinemia (52%) compared with M-LPS cows. Blood urea nitrogen increased after LPS, and the relative change in BUN was elevated in E-LPS cows compared with M-LPS cows (36% and 13%, respectively, from 9 to 24 h). During P1, metabolic flexibility was increased in liver and muscle in early lactating cows compared with mid-lactation cows, but 12 h after LPS, metabolic flexibility was reduced and did not differ by LS. In conclusion, IMM LPS caused severe immune activation, and E-LPS cows had a more intense inflammatory response compared with M-LPS cows, but the effects on milk synthesis was similar between LS. Some parameters of the E-LPS metabolic profile suggest continuation of metabolic adjustments associated with early lactation to support both a robust immune system and milk synthesis.

Effects of heat stress on markers of skeletal muscle proteolysis in dairy cattle.

Heat stress (HS) markedly affects postabsorptive energetics and protein metabolism. Circulating urea nitrogen increases in multiple species during HS and it has been traditionally presumed to stem from increased skeletal muscle proteolysis; however, this has not been empirically established. We hypothesized HS would increase activation of the calpain and proteasome systems as well as increase degradation of autophagosomes in skeletal muscle. To test this hypothesis, lactating dairy cows (~139 d in milk; parity ~2.4) were exposed to thermal neutral (TN) or HS conditions for 7 d (8 cows/environment). To induce HS, cattle were fitted with electric blankets for the duration of the heating period and the semitendinosus was biopsied on d 7. Heat stress increased rectal temperature (1.3°C) and respiratory rate (38 breaths per minute) while it decreased dry matter intake (34%) and milk yield (32%). Plasma urea nitrogen (PUN) peaked following 3 d (46%) and milk urea nitrogen (MUN) peaked following 4 d of environmental treatment and while both decreased thereafter, PUN and MUN remained elevated compared with TN (PUN: 20%; MUN: 27%) on d 7 of HS. Contrary to expectations, calpain I and II abundance and activation and calpain activity were similar between groups. Likewise, relative protein abundance of E3 ligases, muscle atrophy F-box protein/atrogin-1 and muscle ring-finger protein-1, total ubiquitinated proteins, and proteasome activity were similar between environmental treatments. Finally, autophagosome degradation was also unaltered by HS. Counter to our hypothesis, these results suggest skeletal muscle proteolysis is not increased following 7 d of HS and call into question the presumed dogma that elevated skeletal muscle proteolysis, per se, drives increased AA mobilization.

Modeling the effects of heat stress in animal performance and enteric methane emissions in lactating dairy cows.

Heat stress (HS) negatively affects dry matter intake (DMI), milk yield (MY), feed efficiency (FE), and free water intake (FWI) in dairy cows, with detrimental consequences to animal welfare, health, and profitability of dairy farms. Absolute enteric methane (CH4) emission, yield (CH4/DMI), and intensity (CH4/MY) may also be affected. Therefore, the goal of this study was to model the changes in dairy cow productivity, water intake, and absolute CH4 emissions, yield, and intensity with the progression (days of exposure) of a cyclical HS period in lactating dairy cows. Heat stress was induced by increasing the average temperature by 15°C (from 19°C in the thermoneutral period to 34°C) while keeping relative humidity constant at 20% (temperature-humidity index peaks of approximately 83) in climate-controlled chambers for up to 20 d. A database composed of individual records (n = 1,675) of DMI and MY from 82 heat-stressed lactating dairy cows housed in environmental chambers from 6 studies was used. Free water intake was also estimated based on DMI, dry matter, crude protein, sodium, and potassium content of the diets, and ambient temperature. Absolute CH4 emissions was estimated based on DMI, fatty acids, and dietary digestible neutral detergent fiber content of the diets. Generalized additive mixed-effects models were used to describe the relationships of DMI, MY, FE, and absolute CH4 emissions, yield, and intensity with HS. Dry matter intake and absolute CH4 emissions and yield reduced with the progression of HS up to 9 d, when it started to increase again up to 20 d. Milk yield and FE reduced with the progression of HS up to 20 d. Free water intake (kg/d) decreased during the exposure to HS mainly because of a reduction in DMI; however, when expressed in kg/kg of DMI it increased modestly. Methane intensity also reduced initially up to d 5 during HS exposure but then started to increase again following the DMI and MY pattern up to d 20. However, the reductions in CH4 emissions (absolute, yield, and intensity) occurred at the expense of decreases in DMI, MY, and FE, which are not desirable. This study provides quantitative predictions of the changes in animal performance (DMI, MY, FE, FWI) and CH4 emissions (absolute, yield, and intensity) with the progression of HS in lactating dairy cows. The models developed in this study could be used as a tool to help dairy nutritionists to decide when and how to adopt strategies to mitigate the negative effects of HS on animal health and performance and related environmental costs. Thus, more precise and accurate on-farm management decisions could be taken with the use of these models. However, application of the developed models outside of the ranges of temperature-humidity index and period of HS exposure included in this study is not recommended. Also, validation of predictive capacity of the models to predict CH4 emissions and FWI using data from in vivo studies where these variables are measured in heat-stressed lactating dairy cows is required before these models can be used.

Comparison of production-related responses to hyperinsulinemia and hypoglycemia induced by clamp procedures or heat stress of lactating dairy cattle.

Hyperinsulinemia concurrent with hypoglycemia is one of a myriad of physiological changes typically experienced by lactating dairy cows exposed to heat stress, the consequences of which are not yet well defined or understood. Therefore, the objective of this experiment was to separate the production-related effects of hyperinsulinemia with hypoglycemia from those of a hyperthermic environment. Multiparous lactating Holstein cows (n = 23; 58 ± 4 d in milk, 3.1 ± 0.3 lactations) were housed in temperature-controlled rooms and all were subjected to 4 experimental periods as follows: (1) thermoneutral (TN; temperature-humidity index of 65.1 ± 0.2; d 1-5), (2) TN + hyperinsulinemic-hypoglycemic clamp (HHC; insulin infused at 0.3 µg/kg of BW per h, glucose infused to maintain 90 ± 10% of baseline blood glucose for 96 h; d 6-10), (3) heat stress (HS; temperature-humidity index of 72.5 ± 0.2; d 16-20), and (4) HS + euglycemic clamp (EC; glucose infused to reach 100 ± 10% of TN baseline blood glucose for 96 h; d 21-25). Cows were fed and milked twice daily. Feed refusals were collected once daily for calculation of daily dry matter intake, and milk samples were collected at the beginning and end of each period for component analyses. Circulating insulin concentrations were measured in daily blood samples, whereas glucose concentrations were measured more frequently and variably in association with clamp procedures. Rectal temperatures and respiration rates were greater during HS than TN, as expected, and states of hyperinsulinemia and hypoglycemia were successfully induced by the HHC and high ambient temperatures (HS and EC). Feed intake differed based upon thermal environment as it was similar during TN and HHC periods, and declined for HS and EC. Milk production was not entirely reflective of feed intake as it was greatest during TN, intermediate during HHC, and lowest during HS and EC. All milk components differed with the experimental period, primarily in response to the thermal environment. Interestingly, TN baseline glucose concentrations were highly correlated with the change in glucose from TN to HS, and were related to glycemic status during HS. Furthermore, although few in number, those cows that failed to become hypoglycemic during HS tended to have a greater reduction in milk yield. The work presented here addresses a critical knowledge gap by broadening our understanding of the physiological response to heat stress and the related changes in glycemic state. This broadened understanding is fundamental for the development of novel, innovative management strategies as the dairy industry is compelled to become increasingly efficient in spite of global warming.

Evaluating acute inflammation's effects on hepatic triglyceride content in experimentally induced hyperlipidemic dairy cows in late lactation.

Inflammation appears to be a predisposing factor and key component of hepatic steatosis in a variety of species. Objectives were to evaluate effects of inflammation [induced via intravenous lipopolysaccharide (LPS) infusion] on metabolism and liver lipid content in experimentally induced hyperlipidemic lactating cows. Cows (765 ± 32 kg of body weight; 273 ± 35 d in milk) were enrolled in 2 experimental periods (P); during P1 (5 d), baseline data were obtained. At the start of P2 (2 d), cows were assigned to 1 of 2 treatments: (1) intralipid plus control (IL-CON; 3 mL of saline; n = 5) or (2) intralipid plus LPS (IL-LPS; 0.375 µg of LPS/kg of body weight; n = 5). Directly following intravenous bolus (saline or LPS) administration, intralipid (20% fat emulsion) was intravenously infused continuously (200 mL/h) for 16 h to induce hyperlipidemia during which feed was removed. Blood samples were collected at -0.5, 0, 4, 8, 12, 16, 24, and 48 h relative to bolus administration, and liver biopsies were obtained on d 1 of P1 and at 16 and 48 h after the bolus. By experimental design (feed was removed during the first 16 h of d 1), dry matter intake decreased in both treatments on d 1 of P2, but the magnitude of reduction was greater in LPS cows. Dry matter intake of IL-LPS remained decreased on d 2 of P2, whereas IL-CON cows returned to baseline. Milk yield decreased in both treatments during P2, but the extent and duration was longer in LPS-infused cows. Administering LPS increased circulating LPS-binding protein (2-fold) at 8 h after bolus, after which it markedly decreased (84%) below baseline for the remainder of P2. Serum amyloid A concentrations progressively increased throughout P2 in IL-LPS cows (3-fold, relative to controls). Lipid infusion gradually increased nonesterified fatty acids and triglycerides in both treatments relative to baseline (3- and 2.5-fold, respectively). Interestingly, LPS infusion blunted the peak in nonesterified fatty acids, such that concentrations peaked (43%) higher in IL-CON compared with IL-LPS cows and heightened the increase in serum triglycerides (1.5-fold greater relative to controls). Liver fat content remained similar in IL-LPS relative to P1 at 16 h; however, hyperlipidemia alone (IL-CON) increased liver fat (36% relative to P1). No treatment differences in liver fat were observed at 48 h. In IL-LPS cows, circulating insulin increased markedly at 4 h after bolus (2-fold relative to IL-CON), and then gradually decreased during the 16 h of lipid infusion. Inducing inflammation with simultaneous hyperlipidemia altered the characteristic patterns of insulin and LPS-binding protein but did not cause fatty liver.

Skeletal muscle and hepatic insulin signaling is maintained in heat-stressed lactating Holstein cows.

Multiparous cows (n=12; parity=2; 136±8 d in milk, 560±32kg of body weight) housed in climate-controlled chambers were fed a total mixed ration (TMR) consisting primarily of alfalfa hay and steam-flaked corn. During the first experimental period (P1), all 12 cows were housed in thermoneutral conditions (18°C, 20% humidity) with ad libitum intake for 9 d. During the second experimental period (P2), half of the cows were fed for ad libitum intake and subjected to heat-stress conditions [WFHS, n=6; cyclical temperature 31.1 to 38.9°C, 20% humidity: minimum temperature humidity index (THI)=73, maximum THI=80.5], and half of the cows were pair-fed to match the intake of WFHS cows in thermal neutral conditions (TNPF, n=6) for 9 d. Rectal temperature and respiration rate were measured thrice daily at 0430, 1200, and 1630 h. To evaluate muscle and liver insulin responsiveness, biopsies were obtained immediately before and after an insulin tolerance test on the last day of each period. Insulin receptor (IR), insulin receptor substrate 1 (IRS-1), AKT/protein kinase B (AKT), and phosphorylated AKT (p-AKT) were measured by Western blot analyses for both tissues. During P2, WFHS increased rectal temperature and respiration rate by 1.48°C and 2.4-fold, respectively. Heat stress reduced dry matter intake by 8kg/d and, by design, TNPF cows had similar intake reductions. Milk yield was decreased similarly (30%) in WFHS and TNPF cows, and both groups entered into a similar (-4.5 Mcal/d) calculated negative energy balance during P2. Insulin infusion caused a less rapid glucose disposal in P2 compared with P1, but glucose clearance did not differ between environments in P2. In liver, insulin increased p-AKT protein content in each period. Phosphorylation ratio of AKT increased 120% in each period after insulin infusion. In skeletal muscle, protein abundance of the IR, IRS, and AKT remained stable between periods and environment. Insulin increased skeletal muscle p-AKT in each period, but the phosphorylation ratio (abundance of phosphorylated protein:abundance of total protein) of AKT was decreased in P2 for TNPF animals, but not during WFHS. These results indicate that mild systemic insulin resistance during HS may be related to reduced nutrient intake but skeletal muscle and liver insulin signaling remains unchanged.

Prepubertal tamoxifen treatment affects development of heifer reproductive tissues and related signaling pathways.

Prepubertal exposure of the developing ovaries and reproductive tract (RT) to estrogen or xenoestrogens can have acute and long-term consequences that compromise the reproductive performance of cattle. This research examined effects of the selective estrogen receptor modulator tamoxifen (TAM) on gene and protein abundance in prepubertal ovaries and RT, with a particular focus on signaling pathways that affect morphology. Tamoxifen was administered to Holstein heifer calves (n=8) daily (0.3mg/kg subcutaneously) from 28 to 120 d of age, when tissues were collected. Control calves (n=7) received an equal volume of excipient. Weight, gross measurements, and samples of reproductive tissues were collected, and protein and mRNA were extracted from snap-frozen samples of vagina, cervix, uterus, oviduct, ovary, and liver. Neither estradiol nor insulin-like growth factor I (IGFI) concentrations in the serum were affected by TAM treatment. Tamoxifen treatment reduced ovarian weight independently from effects on antral follicle populations, as there was no difference in visible antral follicle numbers on the day of collection. Estrogen receptor α (ESR1) and ß (ESR2) mRNA, ESR1 protein, IGFI, progesterone receptor, total growth hormone receptor, WNT4, WNT5A, and WNT7A mRNA, in addition to mitogen-activated protein kinase (MAPK) and phosphorylated MAPK proteins were affected differently depending on the tissue examined. However, neither IGFI receptor mRNA nor protein abundance were affected by TAM treatment. Results indicate that reproductive development in prepubertal Holstein heifer calves is TAM-sensitive, and that bovine RT and ovarian development are supported, in part, by estrogen receptor-dependent mechanisms during the period studied here. Potential long-term consequences of such developmental disruption remain to be defined.

Effects of acute heat stress on lipid metabolism of bovine primary adipocytes.

Heat stress (HS) affects numerous physiological processes including nutrient partitioning and lipid metabolism. Objectives of this study were to evaluate how acute HS affects lipid metabolism in subcutaneous adipose tissue of dairy cattle. Adipose tissue biopsies were performed on Holstein cows for bovine primary adipocyte isolation and cultured at either 42°C (HS) or 37°C (thermal neutral, TN). Adipocytes were incubated with increasing isoproterenol (ISO), and with increasing concentrations of insulin in the presence of ISO to evaluate changes in lipolysis. Incorporation of radioactive acetate into lipids was measured as an indicator of lipogenesis. Abundance and phosphorylation of several lipolytic and lipogenic proteins were also measured. Adipocytes exposed to HS had an elevated maximal response to ISO and were more sensitive to lipolytic stimulation by ISO compared with cells cultured at TN. Thermal treatment did not affect the antilipolytic effects of insulin in the presence of ISO. Lipogenesis measured as acetate incorporation was not altered by HS, but a temperature by insulin interaction was observed for the regulation of acetyl CoA carboxylase, such that the presence of insulin resulted in a reduction in phosphorylation of acetyl CoA carboxylase in adipocytes cultured at TN but not HS conditions. Results of this study demonstrate that acute HS has a direct effect on the regulation of lipolysis and the rate-limiting enzyme of lipogenesis in isolated bovine adipocytes.

Short communication: Hepatic progesterone-metabolizing enzymes cytochrome P450 2C and 3A in lactating cows during thermoneutral and heat stress conditions.

Two experiments were performed to determine the effects of heat stress (HS) and insulin on hepatic mRNA abundance of enzymes responsible for metabolizing progesterone [cytochrome P450 2C and 3A (CYP2C and CYP3A)]. To distinguish the direct effects of HS from decreased dry matter intake, cohorts were pair fed (PF) in thermoneutral conditions to match the intake of the HS cows during both experiments. In the first experiment, multiparous late-lactation Holstein cows (n=12, 305±33 d in milk) housed in climate-controlled chambers were subjected to 2 experimental periods: (1) thermoneutral (TN) conditions (18°C, 20% humidity) with ad libitum intake (TN and well fed) for 9 d; and (2) either HS conditions (cyclical temperature 31-40°C, 20% humidity) fed for ad libitum intake (n=6), or TN conditions and PF to match the HS animal (n=6) for 9 d. To evaluate hepatic gene expression during experiment 1, biopsies were obtained at the end of each period. In the second experiment, multiparous mid-lactation Holstein cows (n=12, 136±8 DIM) were housed and fed in conditions similar to those described for the first experiment. Liver biopsies were obtained immediately before and after an insulin tolerance test administered on d 6 of each period. No effects of exogenous insulin were observed on any of the tested variables, nor were there interactions between environment (TN/HS or well fed/PF) and insulin administration. Heat stress decreased hepatic CYP2C expression during both experiments. The relative abundance of CYP3A was not affected by environmental conditions in the late-lactation cows (first experiment), but was reduced by HS in the mid-lactation cows (second experiment). Interestingly, during experiment 2, hepatic CYP3A expression also decreased during PF. These results suggest that HS reduces the capacity of the liver to metabolize progesterone through distinct effects on CYP2C and CYP3A, and that the effects appear to vary based upon stage of lactation. Ultimately, HS may affect reproductive outcomes by reducing the abundance of the enzymes responsible for the breakdown of progesterone. This reduction could serve as a beneficial adaptation for rescuing early embryos or may be detrimental, as it affects feedback mechanisms necessary for proper cyclicity.

Hypoxia simultaneously alters satellite cell-mediated angiogenesis and hepatocyte growth factor expression.

Skeletal muscle regeneration is a multifaceted process requiring the spatial and temporal coordination of myogenesis as well as angiogenesis. Hepatocyte growth factor (HGF) plays a pivotal role in myogenesis by activating satellite cells (SC) in regenerating muscle and likely plays a role as a contributor to revascularization. Moreover, repair of a functional blood supply is critical to ameliorate tissue ischemia and restore skeletal muscle function, however effects of hypoxia on satellite cell-mediated angiogenesis remain unclear. The objective of this study was to examine the role of HGF and effect of hypoxia on the capacity of satellite cells to promote angiogenesis. To characterize the role of HGF, a microvascular fragment (MVF) culture model coupled with satellite cell conditioned media (CM) was employed. The activity of HGF was specifically blocked in SC CM reducing sprout length compared to control CM. In contrast, MVF sprout number did not differ between control or HGF-deficient SC CM media. Next, we cultured MVF in the presence of CM from satellite cells exposed to normoxic (20% O2 ) or hypoxic (1% O2 ) conditions. Hypoxic CM recapitulated a MVF angiogenic response identical to HGF deficient satellite cell CM. Hypoxic conditions increased satellite cell HIF-1α protein abundance and VEGF mRNA abundance but decreased HGF mRNA abundance compared to normoxic satellite cells. Consistent with reduced HGF gene expression, HGF promoter activity decreased during hypoxia. Taken together, this data indicates that hypoxic modulation of satellite cell-mediated angiogenesis involves a reduction in satellite cell HGF expression.

Transplantation and perfusion of microvascular fragments in a rodent model of volumetric muscle loss injury.

Few clinical options are available for the treatment of volumetric muscle loss (VML). An important consideration that needs to be addressed for the development of treatments for these injuries is the establishment of a vascular supply sufficient to support skeletal muscle regeneration. The objective of the current study was to evaluate the potential for microvascular fragments (MVFs) harvested from adipose tissue to support tissue perfusion for VML. Tibialis anterior muscle defects in rats were replaced with constructs that were created on the day of surgery containing either (1) collagen only (COL), (2) freshly isolated microvascular fragments in collagen (MVF), or (3) adipose tissue derived stem cells (ASCs) in collagen. Muscles were harvested 7 and 14 days after surgery. Defects treated with MVFs had a vessel density higher than the other groups at both 7 and 14 days, and those treated with ASCs had a higher vessel density than COL by day 14 (p < 0.05). Perfused vessels were observed in both the ASC and MVF treated defects at day 14, as well as at day 7 in the MVF. This study supports the use of MVFs as a platform to improve tissue perfusion to treat large VML defects. The use of freshly isolated MVFs on the day of surgery supports their clinical use and application.

A dose-response evaluation of rumen-protected niacin in thermoneutral or heat-stressed lactating Holstein cows.

Twenty-four multiparous high-producing dairy cows (40.0±1.4kg/d) were used in a factorial design to evaluate effects of 2 environments [thermoneutral (TN) and heat stress (HS)] and a dose range of dietary rumen-protected niacin (RPN; 0, 4, 8, or 12g/d) on body temperature, sweating rate, feed intake, water intake, production parameters, and blood niacin concentrations. Temperature-humidity index values during TN never exceeded 68 (stress threshold), whereas temperature-humidity index values during HS were above 68 for 24h/d. The HS environment increased hair coat and skin, rectal, and vaginal temperatures; respiration rate; skin and hair coat evaporative heat loss; and water intake and decreased DMI (3.5kg/d), milk yield (4.1kg/d), 4% fat-corrected milk (2.7kg/d), and milk protein yield (181.7g/d). Sweating rate increased during HS (12.7g/m(2) per h) compared with TN, but this increase was only 10% of that reported in summer-acclimated cattle. Niacin supplementation did not affect sweating rate, dry-matter intake, or milk yield in either environment. Rumen-protected niacin increased plasma and milk niacin concentrations in a linear manner. Heat stress reduced niacin concentration in whole blood (7.86 vs. 6.89µg/mL) but not in milk. Reduced blood niacin concentration was partially corrected by dietary RPN. An interaction existed between dietary RPN and environment; dietary RPN linearly increased water intake in both environments, but the increase was greater during HS conditions. Increasing dietary RPN did not influence skin temperatures. During TN, supplementing 12g/d of RPN increased hair coat (unshaved skin; 30.3 vs. 31.3°C at 1600h) but not shaved skin (32.8 vs. 32.9°C at 1600h) temperature when compared with 0g/d at all time points, whereas the maximum temperature (18°C) of the room was lower than skin temperature. These data suggest that dietary RPN increased water intake during both TN and HS and hair coat temperature during TN; however, core body temperature was unaffected. Thus, encapsulated niacin did not improve thermotolerance of winter-acclimated lactating dairy cows exposed to moderate thermal stress in Arizona.

The contribution of biological sex to heat stress-mediated outcomes in growing pigs.

Heat stress (HS) negatively impacts a variety of production parameters in growing pigs; however, the impact of biological sex on the HS response is largely unknown. To address this, 48 crossbred barrows and gilts (36.8 ± 3.7 kg BW) were individually housed and assigned to one of three constant environmental conditions: (1) thermoneutral (TN) (20.8 ± 1.6 °C; 62.0 ± 4.7% relative humidity; n = 8/sex), (2) HS (39.4 ± 0.6 °C; 33.7 ± 6.3% relative humidity) for 1 d (HS1; n = 8/sex), or (3) or for 7 d (HS7; n = 8/sex). As expected, HS increased rectal temperature (Tr) following 1 d of HS (1.0 °C; P < 0.0001) and 7 d of HS (0.9 °C; P < 0.0001). By 7 d, heat-stressed gilts were cooler than barrows (0.4 °C; P = 0.016), despite identical heating conditions. There was a main effect of sex such that barrows had higher Tr than gilts (P = 0.031). Heat-stressed pigs on d 1 had marked reductions in feed intake and BW compared to TN (P < 0.0001). One day of HS resulted in negative gain to feed (G:F) in barrows and gilts and was reduced compared to TN (P < 0.0001). Notably, following 1 d of HS, the variability of G:F was greater in gilts than in barrows. Between 1 and 7 d of HS, G:F improved in barrows and gilts and were similar to TN pigs, even though HS barrows had higher Tr than gilts over this period. Heat stress for 1 and 7 d reduced empty gastrointestinal tract weight compared to TN (P < 0.0001). Interestingly, HS7 gilts had decreased gastrointestinal tract weight compared to HS1 gilts (2.43 vs 2.72 kg; P = 0.03), whereas it was similar between HS1 and HS7 barrows. Lastly, a greater proportion of gastrointestinal contents was in the stomach of HS1 pigs compared to TN and HS7 (P < 0.05), which is suggestive of decreased gastric emptying. Overall, HS barrows maintained an elevated Tr compared to HS gilts through the duration of the experiment but also maintained similar growth and production metrics compared to gilts, despite this higher temperature.

Satellite cells isolated from aged or dystrophic muscle exhibit a reduced capacity to promote angiogenesis in vitro.

Deficits in skeletal muscle function exist during aging and muscular dystrophy, and suboptimal function has been related to factors such as atrophy, excessive inflammation and fibrosis. Ineffective muscle regeneration underlies each condition and has been attributed to a deficit in myogenic potential of resident stem cells or satellite cells. In addition to reduced myogenic activity, satellite cells may also lose the ability to communicate with vascular cells for coordination of myogenesis and angiogenesis and restoration of proper muscle function. Objectives of the current study were to determine the angiogenic-promoting capacity of satellite cells from two states characterized by dysfunctional skeletal muscle repair, aging and Duchenne muscular dystrophy. An in vitro culture model composed of satellite cells or their conditioned media and rat adipose tissue microvascular fragments (MVF) was used to examine this relationship. Microvascular fragments cultured in the presence of rat satellite cells from adult muscle donors (9-12 month of age) exhibited greater indices of angiogenesis (endothelial cell sprouting, tubule formation and extensive branching) than MVF co-cultured with satellite cells from aged muscle donors (24 month of age). We sought to determine if the differential degree of angiogenesis we observed in the co-culture setting was due to soluble factors produced by each satellite cell age group. Similar to the co-culture experiment, conditioned media produced by adult satellite cells promoted greater angiogenesis than that of aged satellite cells. Next, we examined differences in angiogenesis-stimulating ability of satellite cells from 12 mo old MDX mice or age-matched wild-type mice. A reduction in angiogenesis activity of media conditioned by satellite cells from dystrophic muscle was observed as compared to healthy muscle. Finally, we found reduced gene expression of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in both aged and dystrophic satellite cells compared to their adult and normal counterparts, respectively. These results indicate that functional deficits in satellite cell activities during aging and diseased muscle may extend to their ability to communicate with other cells in their environment, in this case cells involved in angiogenesis.

Effects of prolonged nutrient restriction on baseline and periprandial plasma ghrelin concentrations of postpubertal Holstein heifers.

Objectives of this study were to measure both daily and periprandial plasma ghrelin concentrations of postpubertal Holstein heifers during prolonged undernutrition. Following an acclimation period, Holstein heifers [n=10; 339.5 ± 8.6 kg of body weight (BW)] were fed ad libitum [well fed (WF); n=5] or restricted to 50% of ad libitum intake [underfed (UF); n=5) for 8 wk. Body condition scores (BCS) were recorded at the beginning and end of the treatment period, and weekly measurements of BW, plasma ghrelin, progesterone, and nonesterified fatty acids (NEFA) concentrations were obtained. Ovarian follicular and luteal structures were measured twice weekly via transrectal ultrasonography. Plasma ghrelin concentrations were also measured during a periprandial window bleed conducted at the end of the experiment. During the window bleed, samples were collected every 15 min between 0500 and 0900 h, with feed offered at 0700 h. Underfed heifers lost BW and BCS, whereas WF heifers gained weight and either increased or maintained BCS. Chronic underfeeding increased circulating ghrelin and NEFA concentrations. By wk 4 of the treatment period, circulating ghrelin concentrations of the UF heifers reached a plateau. Periprandial fluctuations in ghrelin concentrations were apparent as plasma ghrelin concentrations changed over time. Overall differences in periprandial plasma ghrelin concentrations were primarily due to prefeeding effects of plane of nutrition. Plasma ghrelin concentrations and change in BCS were negatively correlated such that heifers that lost the most BCS had the highest concentrations of circulating ghrelin. Two of the 5 UF heifers became anestrus by wk 3 of the treatment period. Despite being of similar age, the heifers that became anestrus had lower BW and plasma ghrelin concentrations than the UF heifers that continued to ovulate. In the current experiment, long-term undernutrition elicited ghrelin responses similar to those reported for shorter durations of nutrient restriction in cattle and other ruminants. These results demonstrate that plane of nutrition is a chronic regulator of plasma ghrelin concentrations, and that these concentrations can be experimentally manipulated in postpubertal heifers for up to 8 wk with no evidence of an adaptive response.

Alterations in expression of gluconeogenic genes during heat stress and exogenous bovine somatotropin administration.

Study objectives were to evaluate hepatic gluconeogenic enzyme gene expression in recombinant bovine somatotropin (rbST)-treated lactating dairy cattle during heat stress (HS) or in thermal-neutral, pair-fed (PF) animals. Twenty-two multiparous (99 d in milk, 656 kg of BW) Holstein cows were subjected to 3 consecutive experimental periods (7 d each): (1) thermal neutral, (2) HS or PF, and (3) HS or PF with rbST (Posilac, administered on d 1 of period 3). Liver biopsies were obtained on the final day of each period. Heat stress conditions progressively decreased dry matter intake for the first 5 to 6 d during period 2 before stabilizing (a decrease of 6.15 kg; 30%) on d 6 and 7, and feed intake remained stable and not different from period 2 during period 3. Cytosolic phosphoenolpyruvate carboxykinase mRNA abundance increased during PF, but was unaffected by HS or bST. Pyruvate carboxylase gene expression increased during HS and PF, and administering bST decreased pyruvate carboxylase mRNA abundance during both HS and PF. Insulin-like growth factor-I gene expression increased following bST administration during HS and PF, confirming hepatic bST responsiveness. Exposure to HS leads to a change in hepatic gluconeogenic enzyme profile that appears to be dependent on plane of nutrition.

Postabsorptive carbohydrate adaptations to heat stress and monensin supplementation in lactating Holstein cows.

Multiparous cows (n=34, 89 d in milk, 537 kg) housed in environmental chambers were fed a control total mixed ration or one containing monensin (450 mg/cow per day) during 2 experimental periods (P): (1) thermal neutral (TN) conditions (constant 20°C) with ad libitum intake for 9 d, and (2) heat stress (HS, n=16) or pair-fed [PF; in TN (PFTN); n=18] for 9 d. Heat-stress was cyclical with temperatures ranging from 29.4 to 38.9°C. Rectal temperatures and respiration rates increased in HS compared with PFTN cows (38.4 to 40.4°C, 40 to 93 breaths/min). Heat stress reduced dry matter intake (DMI, 28%), and by design, PFTN cows had similar intakes. Monensin-fed cows consumed less DMI (1.59 kg/d) independent of environment. Milk yield decreased 29% (9.1 kg) in HS and 15% (4.5 kg) in PFTN cows, indicating that reduced DMI accounted for only 50% of the decreased milk yield during HS. Monensin had no effect on milk yield in either environment. Both HS and PFTN cows entered into calculated negative energy balance (-2.7 Mcal/d), and feeding monensin increased feed efficiency (7%) regardless of environment. The glucose response to an epinephrine (EPI) challenge increased (27%) during P2 for both HS and PFTN cows, whereas the nonesterified fatty acid response to the EPI challenge was larger (56%) during P2 in the PFTN compared with the HS cows. Compared with P1, whole-body glucose rate of appearance (Ra) decreased similarly during P2 in both HS and PFTN cows (646 vs. 514 mmol/h). Although having similar rates of glucose Ra, HS cows synthesized approximately 225 g less milk lactose; therefore, on a milk yield basis, glucose Ra decreased (3.3%) in PFTN but increased (5.6%) in HS cows. Regardless of environment, monensin-fed cows had increased (10%) glucose Ra per unit of DMI. From the results we suggest that the liver remains sensitive but adipose tissue becomes refractory to catabolic signals and that glucose Ra (presumably of hepatic origin) is preferentially utilized for processes other than milk synthesis during HS.

Effects of heat stress on energetic metabolism in lactating Holstein cows.

Heat stress has an enormous economic impact on the global dairy industry, but the mechanisms by which hyperthermia negatively affect systemic physiology and milk synthesis are not clear. Study objectives were to evaluate production parameters and metabolic variables in lactating dairy cows during short-term heat stress or pair-fed conditions coupled with bST administration. Twenty-two multiparous Holstein cows were subjected to 3 experimental periods: 1) thermoneutral conditions with ad libitum intake for 7 d (P1); 2) heat stress (HS) with ad libitum intake (n=10) or pair-fed (PF) in thermoneutral conditions (n=12) for 7 d (P2), and 3) 7 d of HS or PF in conditions as described in P2 with recombinant bovine somatotropin administered on d 1 (P3). All cows received an intravenous glucose tolerance test (GTT) on d 5 of each period. Heat stress conditions were cyclical and temperatures ranged from 29.4 to 38.9 degrees C. Rectal temperatures and respiration rates increased during heat stress (38.6-40.4 degrees C and 44-89 breaths/min, respectively). Heat stress reduced dry matter intake by 30% and by design PF cows had similar intake reductions (28%). During heat stress and pair-feeding, milk yield decreased by 27.6% (9.6kg) and 13.9% (4.8kg), respectively, indicating that reduced feed intake accounted for only 50% of the decreased milk production. Milk yield increased with recombinant bovine somatotropin in both HS (9.7%) and PF (16.1%) cows. Cows in both groups were in positive energy balance (3.95 Mcal/d) during P1 but entered negative energy balance during P2 and P3 (-5.65 Mcal/d). Heat stress and pair-feeding treatments decreased (9.3%) basal glucose concentrations. Heat stress conditions had no effect on basal NEFA levels during P2; however, PF cows (despite a similar calculated energy balance) had a 2-fold increase in basal NEFA concentrations. Both groups had increased plasma urea nitrogen levels during P2 and P3 compared with P1. Basal insulin levels increased (37%) during P2 and P3 in HS cows but did not differ between periods in PF cows. During P2 and compared with P1, PF cows had a decreased rate of glucose disposal, whereas HS cows had a similar disposal rate following the GTT. During P2 and compared with P1, PF cows had a reduced insulin response whereas HS cows had a similar insulin response to the GTT. In summary, reduced nutrient intake accounted for only 50% of heat stress-induced decreases in milk yield, and feed intake-independent shifts in postabsorptive glucose and lipid homeostasis may contribute to the additional reduction in milk yield.

Effects of heat stress and nutrition on lactating Holstein cows: II. Aspects of hepatic growth hormone responsiveness.

Heat stress (HS) is a multibillion-dollar problem for the global dairy industry, and reduced milk yield is the primary contributor to this annual economic loss. Feed intake declines precipitously during HS but accounts for only about 35% of the decreased milk synthesis, indicating that the physiological mechanisms responsible for decreased milk production during HS are only partly understood. Thus, our experimental objectives were to characterize the direct effects of HS on the somatotropic axis, a primary regulator of metabolism and milk yield. We recently reported no differences in mean growth hormone (GH) concentrations, GH pulsatility characteristics, or GH response to growth hormone releasing factor in HS versus pair-fed (PF) thermoneutral controls. Despite similarities in circulating GH characteristics, plasma insulin-like growth factor (IGF)-I concentrations were reduced during heat stress conditions but not in PF animals, suggesting that uncoupling of the hepatic GH-IGF axis may occur during HS. We investigated this possibility by measuring proximal indicators of hepatic GH signaling following a GH bolus. Heat stress but not PF decreased abundance of the GH receptor and GH-dependent signal transducer and activator of transcription (STAT)-5 phosphorylation. Consistent with reduced GH signaling through STAT-5, basal hepatic IGF-I mRNA abundance was lower in HS cows. Thus, the reduced hepatic GH responsiveness (in terms of IGF-I gene expression) observed during HS appears to involve mechanisms at least partially independent of reduced nutrient intake. The physiological significance of reduced hepatic GH receptor abundance during HS is unclear at this time. Aside from reducing IGF-I production, it may reduce other GH-sensitive bioenergetic processes such as gluconeogenesis.