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Journal of Animal Science - Animal Physiology

Intrauterine growth-restricted piglets have similar gastric emptying rates but lower rectal temperatures and altered blood values when compared with normal-weight piglets at birth12

 

This article in JAS

  1. Vol. 94 No. 11, p. 4583-4590
     
    Received: May 14, 2016
    Accepted: July 31, 2016
    Published: October 13, 2016


    4 Corresponding author(s): ca@sund.ku.dk
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doi:10.2527/jas.2016-0639
  1. C. Amdi 4*3,
  2. M. V. Klarlund*33,
  3. J. Hales*,
  4. T. Thymann and
  5. C. F. Hansen*
  1. * Department of Large Animal Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Denmark
     Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Denmark

Abstract

Intrauterine growth-restricted (IUGR) piglets have lower survival rates and are more likely to have empty stomachs 24 h after birth than normal piglets. Although hypoglycemia may result from low colostrum intake per se, it is not known if slow gastric emptying may be an additional risk factor for poor immunization and glucose absorption in IUGR piglets. It is estimated that IUGR piglets consume less colostrum per kilogram BW than normal-weight piglets within the first 24 h, which could be due to a slower gastric emptying rate and a compromised energy metabolism. Therefore, we hypothesized that the gastric emptying rate and blood glucose would be lower in IUGR piglets. We investigated gastric emptying rates in normal and IUGR piglets and blood glucose and rectal temperatures at birth and after 15, 30, 60, and 120 min. In addition, blood parameters relevant for metabolism were studied. Forty-eight piglets (24 normal and 24 IUGR) were classified at birth as either normal or IUGR on the basis of head morphology. Piglets were removed from the sow at birth before suckling, and birth weight was recorded. Pooled porcine colostrum was tube-fed to all piglets at 12 mL/kg BW as soon as possible after birth (t = 0 min). The piglets were randomly allocated to be euthanized at 15, 30, 60, and 120 min (all groups, n = 6) after bolus feeding, and the weights of the stomach and its residuals were recorded. There was no difference in gastric emptying rates between normal and IUGR piglets (P = 0.129); however, gastric DM residuals tended to by greater in IUGR piglets than normal piglets (P = 0.085). Overall, IUGR piglets had lower rectal temperatures (36.2°C ± 0.2°C vs. 37.5°C ± 0.2°C; P < 0.001) and plasma glucose levels (2.8 ± 0.2 vs. 4.1 ± 0.2 mmol; P < 0.001) than normal piglets. Interactions between piglet classification and time were observed in plasma values for NEFA, d-3-hydroxybutyrate, albumin, aspartate, and alanine amino transferase, with greater levels in normal piglets at 15 min (P < 0.05) and 30 min for bile acid (P < 0.05) compared to IUGR piglets. In conclusion, the gastric emptying rates between normal and IUGR piglets were similar, but gastric DM residuals tended to be greater in IUGR piglets. Differences were observed in blood values and rectal temperatures, with lower values in IUGR piglets. Therefore, it is likely that factors like hypothermia and possibly reduced metabolic function are more important during the first hours after birth than gastric retention per se.



INTRODUCTION

Increased litter sizes due to hyperprolific sows have led to litters with up to 30% of piglets being born with signs of intrauterine growth restriction (IUGR; Amdi et al., 2013; Hales et al., 2013), and there is a greater mortality in these piglets in their first few days of life (Hales et al., 2013). Intrauterine growth restriction is usually defined as impaired growth and development of the fetus and/or its organs during gestation (Wu et al., 2006), and because of the brain-sparing effect (Hammond, 1921), IUGR piglets can be recognized by their head shape (Chevaux et al., 2010; Amdi et al., 2013; Hales et al., 2013). It is recommended that piglets need at least 250 g of colostrum within the first 24 h to survive (Quesnel et al., 2012), but IUGR piglets consume only 100 g of colostrum within the first 24 h and less per kilogram of BW than normal-weight piglets (Amdi et al., 2013). The emptying rate of the ventricle of an adult pig follows an exponential curve (Gregory et al., 1990); however, it is not known if normal newborn piglets follow the same curve and if it is different for IUGR piglets. In addition, IUGR piglets have a compromised GI development (Wang et al., 2005), an impaired intestinal nutrient absorption surface (D’Inca et al., 2010), and an increased surface to body mass, making them more susceptible to hypothermia (Herpin et al., 2002). Even though IUGR pigs may consume some colostrum, it is not known if it is retained in the stomach for longer (providing no benefit to the host) or whether it is released to the small intestine, where it may or may not be absorbed.

The aim of this study was to determine if the gastric emptying rates were lower in the IUGR piglets to investigate if this is the reason for a lower colostrum intake. In addition, parameters influencing metabolism were studied. We hypothesized that IUGR piglets would have a lower gastric emptying rate and also lower blood glucose levels and rectal temperatures at birth than normal piglets.


MATERIAL AND METHODS

The experiment was approved by the Danish Animal Experimentation Inspectorate, license number 2014-15-0201-00418. A pilot study was conducted to investigate the approximate amount of colostrum the IUGR piglets could be given per kilogram of BW. Five piglets were tube-fed different amounts ranging from 5 to 15 mL of colostrum and were euthanized after 2 or 3 h. From these preliminary findings, it was decided to tube-feed the piglets with porcine colostrum at a dose of 12 mL/kg BW. A recent study suggests that piglets can be given up to 20 mL/kg BW (Amdi and Hales, 2016).

Animals and Design

The experiment was conducted on a Danish commercial piggery with piglets (Danish Landrace × Danish Yorkshire × Duroc). Forty-eight piglets from 16 litters were classified at birth as either normal or IUGR on the basis of their head morphology (24 normal and 24 IUGR piglets) modified after Hales et al. (2013). Briefly, the piglets were given a visual score at birth from normal to severe IUGR, recognizing the IUGR piglet by a 1) steep dolphin-like forehead, 2) bulging eyes, and 3) hair with no direction of growth. A score of IUGR was given if 2 or 3 of the characteristics were present, and if none of the characteristics were present, the piglets were considered normal and represented control piglets. A visual score was chosen as this is an easier identification method for the farmer than birth weight (see discussion in Amdi et al., 2013). Piglets were removed from the sow before they had suckled and were marked for identification purposes and dried, and the umbilical cord was shortened to 15 cm. When possible, a normal piglet and an IUGR piglet were selected from the same sow, with up to a maximum of 4 piglets selected from the same sow. Birth weight was recorded, and pooled colostrum was previously obtained around farrowing from other sows, warmed to 35°C, and tube-fed (Unomedical feeding tube, Hatting, Horsens, Denmark) to all piglets at 12 mL/kg BW as soon as possible after birth (t = 0 min). Pools of colostrum were distributed evenly among classifications. A sample of the colostrum was obtained for DM analysis. In addition, crown rump length was measured from the crown of the head to the base of the tail (the supine length of the piglet), and gender was noted (normal: 14 males, 9 females; IUGR: 11 males, 14 females). The piglets were randomly allocated to be euthanized at either 15, 30, 60, or 120 min (all groups, n = 6) after bolus feeding. The ambient temperature in the farrowing room was 24°C, and piglets were kept in a boarded-off creep area (with rape granules as bedding) with a 150 W heating lamp providing a temperature range from 24°C to 29°C.

Blood Sampling, Rectal Temperature, and Analyses

Whole blood glucose was measured by a glucose monitor (Accu-Chek Aviva Nano, Roche, Basel, Switzerland). A drop of blood was obtained from the ear of the piglet at birth (before colostrum) and at 15, 30, 60, and 120 min or until the piglets were euthanized. At the same time points rectal temperatures were taken (Apotekets digitaltermometer standard, Apotekets, Hørsholm, Denmark). All piglets were further blood sampled once before euthanasia for plasma by holding the piglets in dorsal recumbency, and 6 mL of blood were drawn from the jugular vein using 22-gauge needles into Vacutainer tubes containing EDTA (BD Vacutainer, Franklin Lakes, NJ). The samples were centrifuged at room temperature for 15 min at 1,200 × g (CM-6MT, Elmi, Riga, Latvia), after which plasma was transferred to Eppendorf tubes (Sarstedt, Nümbrecht, Germany) and immediately frozen at −20°C for later analysis of plasma metabolites (NEFA, creatinine, triglycerides, glucose, d-3-hydroxybutyrate, bile acid, albumin, aspartate amino transferase, alanine amino transferase, blood urea nitrogen, creatinine, lactate, cholesterol, total protein, γ-glutamyl transferase, basic phosphatase, and inorganic phosphate). Plasma analyses were assayed using an Advia 1800 Chemistry System (Siemens Healthcare Diagnostics, Deerfield, IL).

Tissue Sample Collection and DM Analysis

At the end of the experiment, all piglets were anesthetized intramuscularly with a Zoletil mix (Zoletil 50, Virbac, Kolding, Denmark), containing xylacin (Narcoxyl 20mg/mL, MSD Animal Health, Ballerup, Denmark), ketamine (Ketaminol 100 mg/mL, MSD Animal Health, Ballerup, Denmark) and butorphanol (Torbugesic 10mg/mL, ScanVet, Fredensborg, Denmark) and left in an undisturbed covered box with straw to achieve deep anesthesia. After 10 min the piglets were euthanized with an intracardial injection of 2 to 3 mL pentobarbital (200 mg/mL). The stomachs were removed intact and weighed, and subsequently, the contents were removed, weighed, and stored for later DM analysis. The liver, spleen, kidneys, heart, and brain were harvested and blotted dry before they were weighed on a precision scale (Radwag, Radom, Poland). Samples for DM analysis were kept frozen and freeze-dried in a Hetosicc freeze-dryer (Heto, Birkerød, Denmark) at −20°C under vacuum to a constant weight.

Calculations and Statistical Analysis

To evaluate gastric emptying rate, both the DM in the colostrum sample and the DM in the gastric content were calculated. Afterward, the percentage of gastric DM was calculated, and finally, the percentage of gastric emptying was calculated. From these calculations the gastric emptying percentage was plotted against time to obtain a curve of the gastric emptying rate. Data were analyzed univariately (GLM procedure of SAS; SAS Inst. Inc., Cary, NC) according to the following model:where Yij is the dependent variable measured (organ weights, gastric DM residuals, glucose levels, rectal temperatures, blood parameters), μ denotes the overall mean, αi denotes the effect of classification (i = IUGR, normal), βj denotes the effect of time (j = 15, 30, 60, 120 min), (αβ)ij is the interaction between classification and time, and εij describes the random error term. The interaction between classification and time was removed from the final model when it was not significant. Means were separated using the PDIFF option and presented as least squares means ± SEM and were considered significant when P < 0.05 and a tendency when P < 0.10.


RESULTS

Gastric Emptying Rate and Residual DM

Average birth weights were greater in normal piglets than IUGR piglets (1.38 ± 0.04 vs. 0.70 ± 0.04 kg; P < 0.001), and normal piglets were, on average, tube-fed twice as much porcine colostrum as IUGR piglets (17.0 ± 0.5 vs. 8.3 g ± 0.5; P < 0.001). Correspondingly, the total DM content in the colostrum was, on average, nearly double for normal piglets compared to IUGR piglets (4.08 ± 0.12 vs. 2.07 ± 0.12 g; P < 0.001). Figures 1A and 1B show the time course of changes in gastric and DM residuals between normal and IUGR piglets. As expected, there was a clear influence of time (P < 0.001) on the gastric residuals (Fig. 1A), with values declining over time, but no difference between normal and IUGR piglets was observed (P = 0.129). After 120 min the ventricles were almost empty, with 17% and 22% gastric residuals recovered in normal and IUGR piglets. Dry matter residual followed the same pattern as the gastric emptying rate (Fig. 1B), with values declining over time (P < 0.001). Normal piglets tended to have lower DM values than IUGR piglets (P = 0.085).

Figure 1.
Figure 1.

(A) The gastric emptying rate, (B) DM, (C) rectal temperature, and (D) whole blood glucose in normal piglets (triangles) and intrauterine growth-restricted (IUGR) piglets (circles) over the duration of the trial. Values presented are least squares means ± SEM

 

Rectal Temperatures

Overall, IUGR piglets had lower rectal temperatures than normal piglets (36.2°C ± 0.16°C vs. 37.5°C ± 0.17°C; P < 0.001). An initial temperature drop was observed for both normal piglets and IUGR piglets between birth and 15 min, with a subsequent increase in rectal temperature afterward for both normal and IUGR piglets (Fig. 1C; P < 0.001).

Whole Blood Glucose and Composition of Blood Plasma

No differences were found in whole blood glucose levels between normal and IUGR piglets (P = 0.146), but there was an effect of time (P < 0.001; Fig. 1D). For both groups of piglets there was a peak in whole blood glucose between 30 and 60 min after they had been tube-fed colostrum. However, in contrast to whole blood, plasma glucose levels were higher in normal piglets compared to IUGR piglets (4.1 ± 0.2 vs. 2.8 ± 0.2 mmol/L; P < 0.001; Table 1), albeit with no effect of time. There were significant interactions between time and classification for NEFA, d-3-hydroxybutyrate, bile acid, albumin, aspartate amino transferase, and alanine amino transferase, and these are therefore plotted in Fig. 2. Plasma values for NEFA, d-3-hydroxybutyrate, albumin, aspartate, and alanine amino transferase were all increased in normal piglets at 15 min (P < 0.01) and 30 min for bile acid (P < 0.01) compared to IUGR piglets. Plasma parameters with no interactions can be seen in Table 1. There was an effect of time on creatinine levels (P = 0.027), with increasing concentrations over time with greater values at 120 min (169.5 ± 10.9 μmol/L) compared to 15, 30, and 60 min, with values of 125.0 ± 10.4, 125.4 ± 12.1, and 134 ± 0.9 μmol/L, respectively. Normal piglets tended to have greater levels of triglycerides than IUGR piglets (P = 0.057), but triglyceride levels tended to increase over time for both normal and IUGR piglets (P = 0.060).


View Full Table | Close Full ViewTable 1.

Comparison of plasma parameters in normal and IUGR piglets over the duration of the trial1

 
P-value2
Item Normal IUGR SEM Classification Time
n 24 24
Glucose, mmol/L 4.2 2.8 0.2 0.001 0.466
Triglyceride, mmol/L 0.27 0.23 0.01 0.060 0.057
Blood urea nitrogen, mmol/L 3.97 4.04 0.33 0.879 0.827
Creatinine, μmol/L 132.1 145.3 8.0 0.250 0.027
Lactate, mmol/L 4.40 4.93 0.3 0.234 0.175
Cholesterol, mmol/L 1.24 1.35 0.06 0.186 0.449
Total protein, g/L 24.6 25.6 0.5 0.155 0.350
γ-Glutamyl transferase, U/L 53.9 46.7 5.3 0.329 0.836
Basic phosphatase, U/L 1356.6 1618.1 149.5 0.223 0.175
Inorganic phosphate, mmol/L 1.33 1.38 0.06 0.532 0.467
1Values presented are least squares means ± SEM. IUGR = intrauterine growth restricted.
2No interactions between classification and time were observed (P < 0.05).
Figure 2.
Figure 2.

The plasma concentrations of (A) NEFA, (B) d-3-hydroxybutyrate, (C) albumin, (D) bile acid, (E) alanine amino transferase, and (F) aspartate amino transferase in normal piglets (triangles) and IUGR piglets (circles). Normal and intrauterine growth-restricted (IUGR) piglets differed significantly at the specific time point: **P < 0.01, ***P < 0.001. Values presented are least squares means ± SEM.

 

Organ Weights and Ratios

The average organ weights between the classes of piglets are presented in Table 2. The liver, spleen, kidneys, heart, and brain all weighed less in the IUGR piglets (P < 0.001; Table 2). When comparing the weights of the brains, the IUGR piglet’s brain was 2 g less than the normal piglet’s brain, but the relative weight of the brains of the IUGR piglets was 4.5% of BW compared to 2.4% of BW in normal piglets (P < 0.001; Table 2). The relative weight of the kidneys, heart, and stomach was also higher in IUGR piglets than normal piglets (P < 0.001), and there was a tendency for the relative weight of the spleen to be greater in IUGR piglets (P = 0.089). However, the relative weight of the liver was greater in normal piglets (P < 0.01). Further, the brain to liver ratio was 2 times greater in the IUGR piglets compared to normal piglets (P < 0.001).


View Full Table | Close Full ViewTable 2.

Comparison between organ weights and organ indices between normal and IUGR piglets at birth (pooled average over the duration of the trial)1

 
Item Normal IUGR SEM P-value
n 24 24
Piglet characteristics
    Birth weight, g 1,380 700 36 <0.001
    Crown rump length, cm 29.3 24.5 0.3 <0.001
Organ weights, g
    Liver 44.4 20.5 1.5 <0.001
    Spleen 1.4 0.8 0.1 <0.001
    Kidneys 10.1 6.4 0.4 <0.001
    Heart 9.1 5.6 0.3 <0.001
    Brain 32.9 30.7 0.6 0.015
    Stomach 6.7 4.1 0.2 <0.001
Relative organ weights, % BW
    Liver 3.2 2.9 0.1 0.010
    Spleen 0.1 0.1 0.01 0.089
    Kidneys 0.7 0.9 0.04 <0.001
    Heart 0.7 0.8 0.02 <0.001
    Brain 2.4 4.5 0.1 <0.001
    Stomach 0.5 0.6 0.01 <0.001
Ratios
    Brain:heart 3.7 5.7 0.2 <0.001
    Brain:liver 0.8 1.6 0.1 <0.001
1Values presented are least squares means ± SEM. IUGR = intrauterine growth restricted.


DISCUSSION

Intrauterine growth-restricted piglets do not normally have a substantial colostrum intake within the first 24 h (Amdi et al., 2013) to facilitate the required amount for survival (Quesnel et al., 2012). Half the piglets that died from various causes (crushing, weakness, starvation, and others such as congenital defects) between birth and d 1 had empty stomachs (Hales et al., 2013), suggesting that this is one of the main causes for a high piglet mortality in this time period. In addition, this in the main time period for deaths of IUGR piglets (Hales et al., 2013). Although hypoglycemia may result from low colostrum intake per se, it is not known if slow gastric emptying may be an additional risk factor for poor immunization and glucose absorption in IUGR piglets. Our hypothesis that gastric emptying rates would be lower because of a less developed GI in IUGR piglets was only partly confirmed. The interesting findings were that IUGR and normal piglets have a similar gastric emptying, and at 120 min the ventricles are almost empty after a single bolus feeding for both classes of piglets. A tendency was, however, observed in gastric DM residuals with lower values in normal piglets. As expected, IUGR piglets showed greater loss of body temperature, suggesting that heat loss could be as important as colostrum intake. Also, differences were observed in plasma glucose and plasma parameters involved in energy metabolism.

Although gastric emptying rate was the same between normal and IUGR piglets, a tendency for a lower DM retention was observed in the IUGR. This may suggest that the soluble whey fraction of colostrum was passed to the small intestine, whereas the precipitated casein was retained longer in IUGR than normal pigs. Further studies to document the activity of gastric casein-clotting enzymes in IUGR are required. It was also observed that some stomachs contained a small amount of meconium (8 normal and 4 IUGR piglets), and this probably influenced the final DM residual. Gastric secretion and saliva could have altered the gastric emptying pattern, although with results per unit BW this could be assumed to be similar for both classes of piglets. In addition, the composition of a meal, for example, chain length of fatty acids (Hunt and Knox, 1968), AA (Cooke and Moulang, 1972), and caloric density (Siegel et al., 1984), can all influence gastric regulation and emptying. However, the influence of these factors was standardized among pigs as they were given pooled colostrum from the same dams. In addition, although quantities differed between classes, they were given a similar intake per unit of BW.

Blood glucose was similar in normal and IUGR piglets when measured on whole blood. However, once the plasma glucose was analyzed, these showed significantly greater levels in normal piglets compared to IUGR piglets, possibly because of sensitivity of the method (Holtkamp et al., 1975). Although whole blood glucose gives an indication of the general energy state of the animal, only numerical differences were found when analyzed in the present experiment. Plasma glucose is significantly greater than whole blood glucose independent of the method of pretreatment (Holtkamp et al., 1975), and the numerical differences observed in whole blood were thereby amplified in the plasma glucose. This could suggest that IUGR piglets perhaps cannot absorb glucose from the gut at the same rate as normal piglets. This is in accordance with previous findings by Wang et al. (2005), who described a longer and thinner small intestine at birth with reduced villous height in IUGR piglets. Moreover, D’Inca et al. (2010) showed that the intestinal nutrient absorption surface was impaired in IUGR piglets during the first days of life. Another option could be that the IUGR piglet redirects more glucose toward the brain, especially in a situation of hypoxia (Bauer et al., 1998), although no difference was observed in blood lactate values between IUGR and normal piglets, making this explanation less likely. Potential differences in postabsorptive glucose metabolism or use should also be further investigated.

In the current study the overall difference in rectal temperatures between IUGR and normal piglets was more than 1°C, even though they were kept under the same conditions. A decrease in body temperature after birth (postnatal hypothermia) is common in piglets (Malmkvist et al., 2006); however, the extent and duration of postnatal hypothermia is known to correlate negatively with survival chances of the piglet (Tuchscherer et al., 2000). The heated area in a farrowing pen (creep area) cannot compensate for the chilling of the ambient temperature because the piglets prefer to rest near the sow for the first few days of life (Berg et al., 2006), suggesting that additional heat sources might increase the chances of survival in IUGR piglets. In line with these findings, Kammersgaard et al. (2011) found that there was a positive relationship between rectal temperatures at 2 h and birth weight.

Smaller piglets have a lower chance of survival (Tuchscherer et al., 2000). However, IUGR piglets are not just born small as the relative brain weight of IUGR piglets compared to normal piglets does indeed imply that they have been subjected to brain sparing, making them easier to recognize in an on-farm assessment by their head shape rather than only by their birth weight. This is in agreement with previous findings by our group (Amdi et al., 2013) that suggest that if a piglet’s brain weighs ∼3% of total birth weight, then the piglet has not suffered from IUGR during fetal growth. In comparison, the IUGR piglets in this present study had brains weighing ∼4.5% of total BW. All other organs measured except for the liver and spleen also showed greater relative weights in IUGR piglets. The relative liver weights showed that the IUGR piglets have smaller livers compared to normal piglets, and this could potentially influence liver metabolism and capacity. This is in accordance with findings by Chen et al. (2015), who showed that specific enzymes related to liver metabolism were decreased in IUGR piglets. The organ results together suggest that most organs, although fully developed, possibly lack development in maturation, thereby affecting metabolism.

There was no difference in the amount of total protein in the blood between IUGR and normal piglets. Total protein concentrations can be used, among other things, as an indicator of adequacy of quantity and quality of diets, as well as efficiency of utilization of dietary protein in the pig (Štukelj et al., 2010). However, although no difference was found in total protein, there were differences in values of albumin, alanine amino transferase, aspartate amino transferase, NEFA, and d-3-hydroxybutyrate at 15 min after birth. These were all greater in the normal piglets compared to IUGR piglets 15 min after both groups had been tube-fed colostrum. These measured parameters all followed the same pattern, suggesting a deficiency in amino acid metabolism in IUGR piglets and a difference in energy metabolism sources at birth. In agreement, Getty et al. (2015) recently found a compromised ability to extract energy from dietary sources in low-birth-weight piglets (defined as piglets weighing less than 0.9 kg BW) compared to average-birth-weight piglets (1.3 to 1.5 kg BW). In addition, the bile acid levels increased in normal piglets in the 30 min sampling, whereas it first peaked in IUGR piglets after 1 h. Creatine concentrations increased in both classes of piglets during the short duration of the experiment, suggesting an increase in muscle activity in both normal and IUGR piglets. This increase in creatine corresponds to the physiological process at birth where both IUGR and normal piglets maintain body temperature by shivering thermogenesis (Herpin et al., 2002). Normal piglets had greater levels of NEFA at the first blood sampling time compared to IUGR piglets. This indicates that perhaps normal piglets are quicker at using fat from the milk than IUGR piglets. Overall, the plasma parameters measured showed no difference after 1 h; however, it is not known what influence these early differences have on IUGR piglet metabolism, and more research is needed in this area.

In conclusion, IUGR and normal piglets show similar gastric emptying rates. From this notion it seems unlikely that gastric retention per se is the most important risk factor for acute postnatal survival. Instead, other risk factors like hypothermia and possibly reduced metabolic function appear to be more important during the first hours after birth.

 

References

Footnotes


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