Effects of fasting prior to slaughter on pH development and energy metabolism post-mortem in M. longissimus dorsi of pigs

Meat Science 84 (2010) 93–100

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Effects of fasting prior to slaughter on pH development and energy metabolism post-mortem in M. longissimus dorsi of pigs H. Sterten a,d,*, N. Oksbjerg b, T. Frøystein c, A.S. Ekker a, N.P. Kjos d a

Felleskjøpet Fôrutvikling, N-7005 Trondheim, Norway Department of Food Science, Faculty of Agricultural Sciences, University of Aarhus, Blichers Allé 20, P.O. Box 50, DK-Tjele, Denmark c Animalia, Norwegian Meat and Poultry Research Centre, P.O. Box 396, N-0513, Oslo, Norway d Department of Animal and Aquaculture Sciences, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway b

a r t i c l e

i n f o

Article history: Received 8 December 2008 Received in revised form 23 July 2009 Accepted 10 August 2009

Keywords: Pork Fasting Gender Feeding regime Lairage Glycolytic potential pH

a b s t r a c t The aim of this study was to investigate the effect of pre-slaughter fasting time, sex and feeding regime on the development of energy metabolism and pH in M. longissimus dorsi (LD) post-mortem in pigs. Two hundred and seventy pigs of the commercial Norwegian crossbreed Noroc (LYLD) were used involving two sexes (gilts and castrates), two feeding regimes (restricted and ad libitum) and four different fasting treatments: (F4) 4 h fasting, (F175) 17.5 h fasting on the farm, (FO175) 17.5 h fasting overnight at the abattoir, and (FO265) 26.5 h fasting overnight at the abattoir. Additionally the pigs experienced two different abattoir lairage times as fasting treatment F4 and F175 had a lairage time of 1.5 h, while fasting treatment FO175 and FO265 had a lairage time of 23.0 h. A short fasting time of 4 h led to a delayed degradation of glycogen, slow decline in pH and a lower ultimate pH45 h post-mortem (pHu) in the LD compared with a fasting time of 26.5 h which resulted in a rapid breakdown of glycogen and pH decline early post-mortem and a high pHu. Proglycogen was degraded in favour of macroglycogen under anaerobic conditions post-mortem. Feeding the animals in the morning before delivery if slaughtered the same day, results in low pH reduction rate and a low pHu compared with pigs fasted overnight either on farm or at the abattoir. Aiming a higher pHu in LD it should be recommended not to feed the pigs in the morning at the day of slaughter. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction The rate and extent of post-mortem energy metabolism and acidification plays an important role in the conversion of muscle to meat and is important for many different quality properties of fresh pork (Briskey, 1964; Offer & Knight, 1988). Glycolytic potential (GP) is a measure of muscle compounds that can be converted to lactic acid and thus contribute to the post-mortem pH decline (Monin & Sellier, 1985). A controlled reduction of GP in the live animal prior to slaughter might be a potential way to reduce the extent of pH decline post-mortem and thereby improve meat quality traits such as colour and WHC. GP of muscles can be influenced by several factors like genotype (Monin and Sellier, 1985; Enfalt, Lundstrom, Hansson, Lundeheim, & Nystrom, 1997), strategic feeding (Bee, 2002; Rosenvold et al., 2001; Ruusunen, Partanen, Poso, & Puolanne, 2007), rearing conditions (Enfalt et al., 1997), fasting (Bertol, Ellis, Ritter, & McKeith, 2005), and pre-slaughter handling practices (Hambrecht et al., 2004). It can be speculated that the * Corresponding author. Address: Felleskjøpet Fôrutvikling, N-7005 Trondheim, Norway. Tel.: +47 90 64 98 05; fax: +47 73 91 41 34. E-mail address: [email protected] (H. Sterten). 0309-1740/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2009.08.019

feeding regime in the grower – finishing period may have an impact on GP caused by differences in feed intake and furthermore, that male castrates have higher GP due to their higher feed intake compared to female pigs. A fasting period of 16–24 h is recommended in practice (Eikelenboom, Bolink, & Sybesma, 1991) in order to reduce the volume of stomach content and the risk of microbial contamination in the period from delivery to post evisceration. Many studies have showed that a prolonged fasting period before slaughter results in lower muscle glycogen and meat with a higher muscle pHu defined as pH24 h (Eikelenboom et al., 1991; Partanen, Sijander-Rasi, Honkavaara, & Ruusunen, 2007; Warriss & Bevis, 1987; Wittmann, Ecolan, Levasseur, & Fernandez, 1994), while Bidner, Ellis, Witte, Carr, and McKeith (2004) did not find any effect on pHu even after 60 h of fasting. Bertol, Ellis, Ritter, McKeith, and Hamilton (2006) found a reduction in muscle glycogen in pigs fasted 24 h and handled with high intensity, but not in fed pigs with the same high handling intensity. Effect of fasting on glycogen levels in muscle is concluded to be dependent on other factors like feeding and pre-slaughter handling practices (Faucitano et al., 2006; Leheska, Wulf, & Maddock, 2002). Thus, results from studies dealing with the effect of fasting time on pHu are conflicting and furthermore,

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in many of these studies, fasting time is often confounded with resting time at the abattoir. Muscle glycogen exists in two forms; the acid soluble, high molecular weight (107 Da) macroglycogen which is a branched glucose polymer with about 1% protein, and the acid-insoluble, low molecular weight (400,000 Da) proglycogen, which is characterised by a higher protein content of about 10% (Lomako, Lomako, & Whelan, 1991). It is known that the macroglycogen pool increases with high muscle glycogen content (Adamo & Graham, 1998). It has been shown that a decrease in total glycogen induced by feeding a glycogen reducing diet is due to a reduction in the macroglycogen pool only (Rosenvold, Essen-Gustavsson, & Andersen, 2003), and furthermore that proglycogen is degraded in favour of macroglycogen during the first 45–60 min post-mortem leading to rapid pH decline. Consequently, it seems that proglycogen and macroglycogen are different pools with different functions. The objectives of this study were to evaluate the effects of four different fasting treatments prior to slaughter including fasting time and lairage time at the abattoir on glycogen stores in the liver and LD and the breakdown of glycogen including macroglycogen and proglycogen, into intermediate energy metabolites and pH development in M. longissimus dorci (LD) of pigs post-mortem. Moreover, the experimental design made it possible to investigate potential effects of sex and different feeding regimes prior to slaughter on GP and the rate and extent of pH decline post-mortem.

Table 1 The ingredients, calculated and analysed composition of the diet. Item Ingredients (g kg ) Barley Wheat Oats Soy bean meal Peas Animal fat Molasses Limestone Monocalciumphosphate Sodium chloride

2.1. Animals and treatments All pigs were cared for according to the laws and regulations controlling experiments with live animals in Norway (the Animal Protection Act of December 20th, 1974, and the Animal Protection Ordinance concerning experiments with animals of January 15th, 1996). A total of 270 slaughter pigs (135 gilts and 135 castrates) of the commercial Halothane negative Norwegian crossbreed Noroc (Norwegian Landrace  Yorkshire sow and Norwegian Landrace  Duroc boar) were used in the trial. The pigs were allotted 8–10 pigs per pen with the same sex (gilts or castrates) in each pen in a commercial finishing facility. The pigs were fed a commercial grower-finisher diet at two different feeding regimes, restricted twice a day or ad libitum feeding. The composition of the diet is shown in Table 1. The pigs were transported 1.0 h on a commercial truck and slaughtered at a commercial abattoir over a period of 15 days on four slaughter days. During transport and at lairage pigs from different pens were randomly mixed and kept in groups of 8–12 per pen. When fed at lairage the pigs were fed a diet similar to the diet fed in the finishing period. The pigs were stunned with CO2 in a backloader group stunning system (COMI88, Butina APS, Denmark). After stunning the exsanguination, evisceration and chilling of the carcasses followed the standard procedures at the abattoir. The average carcass weight of the pigs was 80.1 kg differing from 55.7 to 102.3 kg. Prior to slaughter the pigs experienced four different fasting treatments (Table 2). The first group was fed at 6.30 in the morning 1.5 h before delivery, transported for 1 h and kept in the lairage until 10:30 am which led to a feed deprivation of 4 h and a lairage time of 1.5 h before slaughter (F4). The second group was not fed in the morning but at 17:00 pm the day before slaughter. Delivery, transport and slaughtering at the same time as in fasting treatment F1 leading to feed deprivation of 17.5 h and a lairage time of 1.5 h (F175). The third group was fed at 6:30 in the morning, transported to the abattoir and kept in the lairage, fed at 14:30 pm and then slaughtered the following day at 8:00 am which led to a feed withdrawal of 17.5 h and a lairage time of

DL-Methionin

393 200 150 100 50 26 15 11 9.2 4.6 0.8

L-Lysine

3.9

L-Threonin

1.5

Acidifier Vitamin–mineral premix

14.3 20.7

Calculated contenta Metabolizable energy (MJ kg 1) Net energy (FEn kg 1)

12.9 1.03

Analyzed content DM (%) Crude protein (g kg 1) DM Crude fat (g kg 1) DM Crude ash (g kg 1) DM a

2. Materials and methods

Grower–finisher diet 1

88.2 183.7 52.2 49.9

Estimated from CVB (2005) tables.

Table 2 Experimental design showing fasting times and abattoir lairage times. Fasting treatment

Fasting time (h)

Abattoir lairage time (h)

F4 F175 FO175 FO265

4.0 17.5 on farm 17.5 overnight at abattoir 26.5 overnight at abattoir

1.5 1.5 23.0 23.0

Additionally each fasting treatment had an equal distribution of the two sexes (gilts and castrates) and the two feeding regimes (restricted and ad libitum).

23.0 h (FO175). The fourth group was fed at 6:30 in the morning, transported to the abattoir and kept overnight in the lairage without any feeding and slaughtered the following day at 8:00 am which led to a feed withdrawal of 26.5 h and a lairage time of 23.0 h (FO265). Thus a 22 4 factorial design was used for this study, which involved two sexes (gilts and castrates), two feeding regimes (restricted and ad libitum) and four different fasting treatments (F4, F175, FO175 or FO265). Additionally the pigs experienced two different abattoir lairage times (time from unloading to slaughter). Eight pens of pigs were assigned to each fasting treatment. Pens were assigned, so there was an equal distribution of feeding regime and sex classes in each treatment. The experimental design and prolonged feed withdrawal exceeding the max. 18 h given in §12 in ‘‘Directive on animal welfare in abattoirs” was approved by the ‘‘The National Animal Research Authority”.

2.2. Meat quality and biochemical measurements 2.2.1. pH measurements pH measurements were made in the centre of the LD at the last rib 5 cm off the midline with a Knick Portamess 751 Calimatic pH meter (Berlin, Germany) attached to a Mettler-Toledo InLabÒ 427 insertion glass electrode (Mettler–Toledo, GmbH, Hackacker, Germany) at the following times post-mortem: 20 min (0.3 h), 2, 8, 20 and 45 h.

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2.2.2. Sampling procedures and biochemical measurements At 15 min post-mortem two samples of the liver (right central lobe) were excised, put in vials and frozen in liquid nitrogen and kept frozen at 80 °C until analysis for glycogen and free glucose content. Muscle samples were taken from LD at the last rib of the right half of the carcass at 2, 8, 20 and 45 h post-mortem, put in vials and frozen in liquid nitrogen and stored at 80 °C until analysis for content of glycolytic components. The liver samples were analysed for glycogen and free glucose content while the muscle samples were analysed for glycogen, glucose-6-phosphate (G-6-P) and lactate content at each sampling time. In addition the muscle samples taken at 2 h post-mortem were analysed for proglycogen, macroglycogen and glucose. Glycogen content of both liver tissue and muscle samples were determined in 25 mg samples, heated in a test tube with 2.5 ml of 1 M HCl at 100 °C for 2 h in sealed glass tubes to hydrolyse the glycogen to glucose units and then centrifuged at 1500 g for 10 min at 4 °C. Twenty-five milli grams samples for determination of proglycogen and macroglycogen were separated as described by Adamo and Graham (1998). In short, the proglycogen was precipitated by perchloric acid. After centrifugation proglycogen in the precipitate and macroglycogen in the supernatant were hydrolysed for 2 h in 1 M HCl. Glucose from the hydrolysed homogenates were analysed according to the same procedure described earlier. G-6-P was measured on muscle homogenates as described by Passonneau and Lowry (1993). In short, G-6-P was oxidized and NADP + reduced to glucolactone-6-phosphate and NADPH, respectively, by the enzyme glucose-6-phosphate dehydrogenase. Glucose-6phosphate is spontaneously hydrolysed to gluconate-6-phosphate. The absorbance of NADPH, which is directly related to the amount of glucose in the samples, was measured at 340 nm. Lactate determination was done by incubation of 25 mg muscle sample on ice vials containing 600 ll of 3 M perchloric acid. The extraction procedure was stopped by adding 1000 ll of 2 M KHCO3 to the vials, and the samples were centrifuged using the same procedure as described earlier. Both glucose and lactate were measured spectrophotometrically as outlined by Passonneau and Lowry (1993) using an Alcyon 300/ISE auto-analyser (Alcyon Analyzer, France). GP was calculated according to the formula proposed by Monin and Sellier (1985): GP = 2([glycogen] + [glucose] + [glucose-6phosphate]) + [lactate], and expressed as micromoles of lactate equivalents per gram of fresh tissue. 2.2.3. Statistical analysis The statistical analysis was carried out with Statistical Analysis System version 8.02 (SAS Institute, Cary, USA). The glycogen and free glucose content in the liver 0.3 h post-mortem, glycogen and other energy metabolites in LD at 2, 8, 20 and 45 h post-mortem, total glycogen, proglycogen, macroglycogen, glucose, G-6-P, lactate and GP in LD 2 h post-mortem, and pH in LD 0.3, 2, 8, 20 and 45 h post-mortem was analysed using the MIXED procedure with fasting treatment, feeding regime, sex and their two-way interactions as fixed factors. Pen was included in the model as a RANDOM factor. Due to significant correlation between total glycogen content in LD

and carcass weight (r = 0.18 and P < 0.01), carcass weight was included as covariate in the model when analysing content of glycogen and free glucose in liver and glycogen and other energy metabolites in LD. Least square means were evaluated using the PDIFF option, and were considered significantly different if P 6 0.05 and as a tendency if P > 0.05 and P 6 0.10. The PROC CORR option was used to calculate the linear correlation between content of glucose residues in liver at 0.3 h post-mortem and GP in LD 2 h post-mortem, GP in LD 2 h post-mortem and pH 20 h and 45 h post-mortem, proglycogen and total glycogen 2 h post-mortem in LD as well as macroglycogen and total glycogen 2 h post-mortem in LD. 3. Results 3.1. Glycogen and free glucose content in liver The analysed content of glucose residues (equivalent to glycogen + free glucose) in the liver 0.3 h post-mortem decreased significantly (P < 0.001) by increasing fasting time from fasting treatment F4 to fasting treatment FO265 (Table 3). The pigs that had experienced fasting treatment F4 had more than twice the content of glucose residues compared to the pigs that had experienced fasting treatment F175, 163.3 and 76.3 lmol/g, respectively. Fasting treatment FO175 resulted in a significant lower content of glucose residues compared with pigs on fasting treatment F175. Fasting treatment FO265 resulted in the lowest content of glucose residues in the liver, although not significantly lower than pigs in fasting treatment FO175. There was a significant interaction between feeding regime and sex (P < 0.05) in the way that castrates on ad libitum feeding tended to have higher liver content of glucose residues than castrates on restricted feeding, 89.0 and 70.4 lmol/g, respectively (P = 0.06). The opposite was true for the gilts where the content of glucose residues were 82.0 and 73.9 lmol/g, after feeding restrictively and ad libitum, respectively, although this difference was not significant (P = 0.36). There were no significant correlations detected between liver glucose residues and GP in LD. No significant correlations between content of liver glucose residues and glycogen content of LD 2 h post-mortem were found with exception of fasting treatment F175 (r = 0.36 and P < 0.01). 3.2. Glycogen and other energy metabolites in M. longissimus dorsi The analysed content of total glycogen in LD during the postmortem period separated on sexes is presented in Table 4. In castrates the glycogen content in LD 2 h post-mortem was significantly lower in fasting treatment FO265 compared with all the other fasting treatments (P < 0.01). Also in gilts the glycogen content at the same measurement time tended to be lower in fasting treatment FO265 compared with the other fasting treatments (P = 0.06). The reduction in glycogen content from 2 to 8 h post-mortem in gilts on fasting treatment F175 and FO175 was significantly higher compared with gilts on fasting treatment F4 (P < 0.05). In castrates the reduction in glycogen content in the same time frame tended

Table 3 LSmeans and SEMa for sum of total glycogen and free glucose content (lmol/g) in the liver measured 0.25 h post-mortem. Fasting treatment

Total glycogen and free glucose

Gilts

Castrates

F4

F175

FO175

FO265

SEM

Pvalue

Restricted

Ad. lib.

SEM

Pvalue

Restricted

Ad. lib.

SEM

Pvalue

163.0 a

76.1b

44.4 c

32.2 c

5.8

<0.001

82.0

73.9

6.0

0.36

70.4

89.0

6.0

0.06

Different letters within a row indicates significant (P < 0.05) difference between values. a Standard error of the mean.

Sex  Feeding regime P-value <0.05

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Table 4 LSmeans and SEMa for content of total glycogen (lmol/g) in M. longissimus dorsi during the post-mortem period separated on sexes. Time post-mortem (h)

Fasting treatment

Feeding regime

F4

F175

FO175

FO265

SEM

P-value

Restricted

Ad. lib.

SEM

P-value

Gilts 2 8 20 45

60.5 44.7 a 27.9 22.2

55.8 32.6 b 23.1 20.3

57.3 28.2 b 21.0 19.3

49.8 27.2 b 25.4 21.5

2.3 2.7 1.5 1.4

0.06 <0.01 0.06 0.56

54.0 33.6 24.7 20.4

57.7 32.7 24.0 21.3

1.6 1.9 1.1 1.0

0.15 0.76 0.65 0.53

Castrates 2 8 20 45

61.3 a 38.4 a 27.1 21.6 ab

59.5 a 32.2 ab 24.7 25.8 a

57.6 a 28.9 b 21.7 20.0 b

40.2 b 25.4 b 23.0 17.9 b

2.7 2.5 1.5 1.4

<0.01 <0.05 0.13 <0.05

57.1 32.9 26.0 a 22.7 a

52.2 29.8 22.3 b 19.5 b

1.7 1.4 1.1 0.9

0.10 0.27 <0.05 <0.05

Different letters within a row indicates significant (P < 0.05) difference between values. a Standard error of the mean.

to be larger in fasting treatments F175 and FO175 than in fasting treatments F4 and FO265 (P = 0.054). Glycogen content in gilts measured 20 h post-mortem tended to be higher in fasting treatment F4 compared to all other treatments (P = 0.06), while no differences were seen between fasting treatments in castrates. The reduction in glycogen levels in the time frame from 8 to 20 h post-mortem was significantly larger in fasting treatment F4 compared to the other fasting treatments in both sexes (P < 0.001). There was a significant interaction between sex and feeding regime on the analysed content of total glycogen in LD during the postmortem period (P < 0.001). The glycogen content in LD of castrates on restricted feeding was significantly higher than when fed ad libitum at the measurement times 20 and 45 h post-mortem, while gilts were not affected by feeding regime. The analysed content of G-6-P and lactate in LD during the postmortem period is presented in Table 5. The initial content of G-6-P in LD measured 2 h post-mortem was significantly lower in fasting treatments F4 and F175 compared with fasting treatments FO175 and FO265 (P < 0.001). During the period from 2 to 20 h post-mortem the different treatments resulted in a different development of G-6-P content in LD. Fasting treatments F4 and F175 resulted in significantly lower levels of G-6-P than fasting treatments FO175 and FO265 at 2 h post-mortem, while at 20 h post-mortem fasting treatment F4 and F175 resulted in significantly higher levels of G-6-P than fasting treatment FO175. No effect of sex was detected on the content of G-6-P in LD. Feeding regime prior to slaughter did not affect the content of G-6-P in LD except at 2 h post-mortem where we found significantly higher content of G-6-P in pigs that had been fed restricted (1.95 lmol/g) compared to ad libitum feeding (1.26 lmol/g) (P < 0.05). The lactate content of LD at 2 h post-mortem was numerically the highest in pigs that had experienced fasting treatment FO175

and lowest in fasting treatment F4. During the period from 2 to 20 h post-mortem the different treatments tended to result in different development of lactate content in LD. The trend is that LD of pigs that had experienced fasting treatment F4 had the lowest muscle lactate content at 2 h post-mortem, but the highest at 20 h post-mortem. The other fasting treatments resulted in more or less similar increase in muscle lactate content in the post-mortem period. At 8 h post-mortem the castrates had a higher muscle lactate content than the gilts (P < 0.01) representing a sex effect. Also at 20 h post-mortem castrates tended to show higher lactate content in LD than in gilts (P = 0.06). Feeding regime seemed to have no effect on muscle lactate content. 3.3. Proglycogen, macroglycogen and glycolytic potential (GP) The main effects of fasting treatments on content of proglycogen, macroglycogen, free glucose and calculated glycolytic potential (GP) in LD 2 h post-mortem are shown in Table 6. The content of proglycogen in LD was significantly higher in pigs that had experienced fasting treatment F4 compared with the other fasting treatments (P < 0.001). The pigs on fasting treatment FO265 had the lowest content of proglycogen, while no differences were seen between fasting treatments F175 and FO175. The content of macroglycogen was significantly higher in fasting treatments F175 and FO175 than in fasting treatments F4 and FO265 (P < 0.001). No significant differences were detected between fasting treatments F4 and FO265, and between fasting treatments F175 and FO175. Both proglycogen and macroglycogen were strongly correlated with total glycogen (P < 0.001) (Table 7). The correlation coefficients between proglycogen and total glycogen were numerically higher than between macroglycogen and total glycogen for all fasting treatments except fasting treatment F175.

Table 5 LSmeans and SEMa for content of glucose-6-phosphate and lactate (lmol/g) in M. longissimus dorsi during the post-mortem period. Time post-mortem (h)

Fasting treatment F4

F175

FO175

FO265

SEM

P-value

Glucose-6-phosphate 2 8 20 45

0.80 b 5.90 12.5 ab 11.3 b

0.36 b 5.65 13.6 a 13.0 a

2.97 a 6.80 10.6 c 10.2 b

2.15 a 6.61 11.4 bc 10.9 b

0.36 0.45 0.4 0.5

<0.001 0.23 <0.001 <0.01

Lactate 2 8 20 45

52.9 ± 2.4 b 82.9 ± 2.1 ab 128.0 ± 2.9 134.0 ± 3.2

56.8 ± 2.4 ab 79.1 ± 2.0 b 118.5 ± 2.8 131.5 ± 3.0

62.9 ± 2.4 a 82.4 ± 2.1 ab 117.3 ± 2.9 130.1 ± 3.1

59.9 ± 2.4 b 88.8 ± 2.1 a 121.9 ± 2.0 130.6 ± 3.2

2.4 2.1 2.8 3.1

<0.05 <0.05 0.06 0.83

Different letters within a row indicates significant (P < 0.05) difference between values. a Standard error of the mean.

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H. Sterten et al. / Meat Science 84 (2010) 93–100 Table 6 LSmeans and SEMa for proglycogen, macroglycogen, glucose content (lmol/g) and glycolytic potential (GP) in M. longissimus dorsi measured two hours post-mortem. Energy metabolites

Proglycogen Macroglycogen Glucose Glycolytic potential

Fasting treatment F4

F175

FO175

FO265

SEM

Pvalue

45.6 a 15.4 b 2.43 b 181.2 ab

33.9 bc 23.9 a 2.44 b 178.1 b

36.1 b 21.9 a 3.58 a 191.1 a

30.6 c 14.7 b 2.65 b 160.2 c

1.4 1.5 0.3 3.8

<0.001 <0.001 <0.05 <0.001

Different letters within a row indicates significant (P < 0.05) difference between values. a Standard error of the mean.

Table 7 Pearson r-values with P-values below (r and P) in LD 2 h post-mortem between content of total glycogen and proglycogen and macroglycogen, and between GP and pH20 h and pH45 h for the different fasting treatments.

Total glycogen Proglycogen Macroglycogen GP pH20

h

pH45

h

F4

F175

FO175

FO265

0.80 <0.001 0.51 <0.001

0.85 <0.001 0.89 <0.001

0.91 <0.001 0.72 <0.001

0.92 <0.001 0.86 <0.001

0.27 <0.05 0.36 <0.01

0.21 0.09 0.50 <0.001

0.28 <0.05 0.32 <0.01

0.66 <0.001 0.52 <0.001

treatments F4 and F175. Especially fasting treatment FO265 shows an extremely rapid pH decline from 0.3 to 2 h post-mortem compared to the other fasting treatments. Comparing 8 and 20 h post-mortem, there seems to be a change in pH levels between fasting treatments. Fasting treatment F4 gave at 8 h post-mortem the highest pH value, while at 20 h post-mortem these pigs were found to have significantly lower muscle pH than both fasting treatments FO175 and FO265. The correlations between GP and pH20 h and pH45 h are shown in Table 7. Significant negative correlations were found between GP and pH20 h for all the fasting treatments except for fasting treatment F175. The correlation between GP and pH45 h were negative and significant for all fasting treatments. The strongest correlations were found in fasting treatment FO265 for both GP and pH20 h and GP and pH45 h. The pH in LD of castrates was significantly lower than in gilts at 2, 8 and 20 h post-mortem, P < 0.01, P < 0.01 and P < 0.05, respectively. The most pronounced difference in pH development between gilts and castrates was observed in fasting treatment FO265 at the early stage post-mortem (Fig. 1). Castrates were found to have a drop in LD pH of 0.42 units from 0.3 to 2 h post-mortem, while the gilts showed a pH drop of only 0.25 units. Also at 8 h post-mortem, in pigs that had experienced fasting treatment FO265, castrates had a lower pH than the gilts. A significant interaction between fasting treatment and sex was detected for pH2 h and pH45 h, P = 0.047 and P = 0.041, respectively. Thus, fasting treatment FO265 showed lower pH2 h compared with the other fasting treatments in castrates, but not in gilts. Moreover, fasting treatment F4 showed lower pH45 h in gilts, but not in castrates. No effect of feeding regime was detected.

GP in the LD was found to be significantly lower in fasting treatment FO265 compared to the other fasting treatments (P < 0.001). The GP in LD of pigs on fasting treatment FO175 was significantly higher than in fasting treatment F175 and FO265, but not significantly different from fasting treatment F4. There were no effects of sex or feeding regime with regard to proglycogen, macroglycogen and GP.

6.8 Gilts Castrates

6.6 6.4

a

pH

6.2

3.4. pH development post-mortem

a

b 6.0

The course of pH in LD during the post-mortem period is presented in Table 8. The different fasting treatments did not influence the initial muscle pH measured 0.3 h post-mortem (P = 0.30), while at all the other measurement times significant differences in pH were observed between the different fasting treatments. Especially at 2 and 8 h post-mortem large differences in muscle pH between the different fasting treatments were found. The pH in LD during the post-mortem period revealed different developments when comparing the different fasting treatments. The pH decline in LD from 0.3 to 8 h post-mortem of pigs that had experienced fasting treatments FO175 and FO265 was more pronounced than in fasting

b 5.8 5.6 5.4 0

5

10

15

20

25 40

45

50

Time (h) Fig. 1. pH in castrates and gilts at fasting treatment FO265 (26.5 h fast overnight at abattoir) from 0.3 to 45 h post-mortem, LSmeans and SEM. Different letters for the different sexes indicates significant differences (P < 0.05).

Table 8 LSmeans and SEMa for pH in M. longissimus dorsi during the post-mortem period. Time post-mortem (h)

Fasting treatment F4

0.3 2 8 20 45

6.59 6.52 6.09 5.61 5.50

Sex

F175 a a b c

6.53 6.50 6.02 5.58 5.55

ab ab c b

FO175

FO265

SEM

P-value

Gilts

Castrates

SEM

P-value

6.54 6.42 5.94 5.66 5.56

6.54 6.20 6.01 5.67 5.58

0.03 0.03 0.03 0.01 0.01

0.30 <0.001 <0.01 <0.001 <0.001

6.57 6.47 a 6.06 a 5.64 a 5.55

6.53 6.36 b 5.97 b 5.62 b 5.54

0.02 0.02 0.02 0.01 0.01

0.14 <0.01 <0.01 <0.05 0.16

b b a ab

c ab a a

Different letters within a row indicates significant (P < 0.05) difference between values. a Standard error of the mean.

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4. Discussion 4.1. Glycogen stores at slaughter The liver glycogen content is a very good trait for evaluating the nutritional status of the animals. In spite of a generally very low content of glycogen in the liver in all the fasting treatments, we found a pronounced decrease in the content of liver glycogen by increasing fasting time. The significant difference in liver glycogen contents between fasting treatments F175 and FO175 exposed to the same fasting time, can be explained by a presumable concomitant reduction in feed intake during lairage before onset of the fasting period in fasting treatment FO175. This very clear decrease in liver glycogen stores by increased feed withdrawal time, is in agreement with earlier findings (Brown, Knowles, Edwards, & Warriss, 1999; Warriss & Brown, 1983). The same authors conclude that the liver glycogen stores are almost completely depleted within the first 18 h of fast which supports the results of the current study. Feeding the pigs during lairage is recommended not only for welfare reasons. Feeding pigs at lairage reduces negative effects on carcass yield (Warriss, 1996), gives less aggressiveness and fighting causing skin damages (Murray, Robertson, Nattress, & Fortin, 2001;Turgeon & Bergeron, 2000), and lower the incidences DFD (dark, firm, and dry) meat due to glycogen exhaustion (Eikelenboom et al., 1991). Warriss, Bevis, and Ekins (1989) demonstrated a positive relationship between liver glycogen and muscle glycogen on feed withdrawal. In our experiment we did not find any substantial relationship between liver glucose residues and the content of glycogen in LD due to weak correlations differing from negative to positive randomly between the fasting treatments. However, we detected significant effects of prolonged fasting time on GP in LD at slaughter which is in accordance with several former studies (Bertol et al., 2005; Jones, Rompala, & Haworth, 1985; Leheska et al., 2002; Warriss et al., 1989). The most pronounced effect on GP, was seen in pigs fasted 26.5 h at lairage. Compared with the 4.0 h fasted pigs and the 17.5 h at lairage fasted pigs, the GP was respectively 13.1% and 19.3% lower in the 26.5 h fasted pigs. Interestingly, we saw that resting time at lairage might have an impact on GP as indicated by the significantly higher GP in fasting treatment FO175 than in fasting treatment F175. The main difference between these two treatments is the prolonged lairage time in fasting treatment FO175 of 23.0 h compared to 1.5 h lairage in fasting treatment F175. During lairage the pigs are exposed to various stress factors like unfamiliar environment and mixing with unfamiliar pigs, leading to fighting and increased muscular activity resulting in a depletion of glycogen stores (Fernandez, Magard, & Tornberg, 1992). However, lairage is not a stress factor itself but is intended to be a recovering from stress associated with transportation. The significant higher GP in the pigs that had experienced a prolonged lairage time but the same fasting time (FO175 compared to F175), indicates that the pigs actually rested and regained metabolic balance during lairage. Replenishment of muscle glycogen by mobilisation of liver glycogen might be an explanation, a theory which is also supported by the lower level of glycogen in the liver of animals that were kept the longest time at lairage. When looking at the two types of glycogen comprising total glycogen we found that the content of proglycogen was significantly higher in pigs on fasting treatment F4 compared to the other fasting treatments, while the pigs that had experienced fasting treatment FO265 had the lowest content of proglycogen. Proglycogen is degraded in favour of macroglycogen under anaerobic conditions post-mortem (Rosenvold et al., 2003; Yla-Ajos et al., 2007) while macroglycogen is reported to be degraded during aerobic low intensity exercise (Essen-Gustavsson, Jensen-Waern, Jonasson, &

Andersson, 2005). These findings support the results from our study where the relationship between total glycogen content and proglycogen content was found to be stronger than between total glycogen and macroglycogen. In the present study, the analytical method for measuring the macroglycogen fraction also included G-6-P, although this fact does not explain the differences observed in macroglycogen contents between the fasting treatments. It is likely that ante-mortem conditions other than fasting time and lairage time might have had an impact on the macroglycogen levels. 4.2. Glycogen degradation post-mortem In the present study we found interesting changes in energy metabolite contents in LD during the post-mortem period from 2 to 45 h, in relation to different pre-slaughter fasting times and lairage times. Besides a lower level of glycogen content at 2 h postmortem in the 26.5 h fasted pigs compared to the pigs that had experienced shorter fasting time, the glycogen degradation seems to be more or less completed already at 8 h post-mortem. In contrast, the F4 pigs expressed a prolonged high glycogen level and had even higher levels at 20 h post-mortem than the F265 pigs at 8 h post-mortem. Looking at the glycogen decline it seems to be more rapid in the early stage post-mortem in pigs fasted at lairage 17.5 and 26.5 h compared to pigs that had experienced either the shortest fasting time or short lairage time and on farm fasting. During the period 2 to 20 h post-mortem the different fasting treatments resulted in different development of G-6-P content in LD. The on farm feed withdrawal and short lairage time resulted in significantly lower levels of G-6-P at 2 h post-mortem compared to feed withdrawal at the abattoir and a long lairage time, while at 20 h post-mortem this situation had changed to the opposite. The content of G-6-P gives an indication of the rate and the stage of the ongoing glycolysis in the LD muscle. Rapid rates of post-mortem glycolysis are associated with low levels of glycogen and high levels of G-6-P, indicative of increased phosphorylase activity (Briskey, 1964; Kastenschmidt, Hoekstra, & Briskey, 1968; Moesgaard, Quistorff, Christensen, Therkelsen, & Jorgensen, 1995). In muscles exhibiting normal rates of glycolysis, G-6-P levels tend to decrease during the first hour post-mortem and increase thereafter indicating an imbalance between glycogenolysis and glycolysis (Hammelman et al., 2003; Kastenschmidt et al., 1968). Comparing the development in LD glycogen and G-6-P contents in the same time frame, we see a tendency of negative correlation in the way that high LD glycogen content corresponds with low G-6-P contents. These findings of glycogen and G-6-P content development in LD post-mortem indicate that there is a delay in the glycogenolysis in pigs that had experienced either short fasting time or on farm feed withdrawal. The explanation for this can be the fasting induced depletion of the energy metabolite creatine phosphate (CP) in the longest fasted pigs resulting in an early onset of the glycogenolysis, while the shortest fasted pigs still have some CP available at the time of slaughter resulting in a delay in the glycogenolysis. CP is an intermediate metabolite, which is rapidly depleted early post-mortem. When CP is present in contents above 3 mmol/kg, ATP levels are kept constant, and no breakdown or even an increase in glycogen may be observed (Henckel & Karlsson, 1997). 4.3. pH development With the subsequent degradation of glycogen, lactate and H+ is accumulated causing a decline in pH. In our experiment the different treatments resulted in a tendency of different development of lactate content in the way that pigs that had experienced the shortest fasting time had the lowest muscle lactate content at 2 h

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post-mortem, but the highest level at 20 h post-mortem. The longest fasted pigs had relative high initial lactate content, while at 20 h post-mortem these had lower lactate content than the shortest fasted pigs. Comparing the development in lactate content with the decline in pH, we see that the delay in lactate accumulation in the shortest fasted pigs corresponds with the delay in pH decline. Surprisingly the very low pH at 2 h post-mortem in the pigs fasted 26.5 h, does not correspond consistently with the lactate levels at the same time of measurement. No obvious explanation is found for this. The relationship between glycogen stores and pHu is reported to be highly correlated but only when the glycogen levels are low (Henckel, Karlsson, & Oksbjerg, 1998; Howard, 1963). Henckel, Karlsson, Jensen, Oksbjerg, and Petersen (2002) showed that the glycogen content at the time of slaughter should be decreased to levels below 53 lmol/g to have a considerable effect on pHu. In the current study we did not measure glycogen at the time of slaughter. Nevertheless, the relationship between GP and pH20 h and pH45 h in our study were significantly negative and moderate to strong in the range from 0.21 to 0.66. This is supported by the study of Hamilton, Miller, Ellis, McKeith, and Wilson (2003) who found a linear relationship between GP and ultimate pH. 4.4. Sex and feeding regime Sex and feeding regime showed interactions regarding glycogen content both in liver and LD, and interactions were also seen between fasting treatment and sex on pH in LD. A higher liver glycogen content in castrates on ad libitum feeding and a corresponding lower glycogen content in LD can be explained by the fact that castrates have a higher feed intake than gilts, and additionally have a more active behaviour leading to an increased depletion in muscle glycogen stores at the time of harvest. Sex differences in aggressive behaviour and fighting following mixing have been shown to have a decisive influence of glycogen depletion, the difference being most pronounced between boars and non-boars (Warriss, 1984). No documentation is found on differences between gilts and castrates with regard to behaviour and glycogen expenditure. pH in the LD of castrates was lower than in gilts and the most pronounced difference was observed in the 26.5 h fasted pigs at the early stage post-mortem. The lower pH in castrates than in gilts in the present study, is not consistent with the study of Eikelenboom et al. (1991) who found a lower pHu in gilts than in castrates. Also D’Souza and Mullan (2002) found lower loin muscle pHu in gilts than in castrates while Hamilton et al. (2002) found no effects of sex on pHu. 4.5. Conclusions The results from this experiment show that the muscle glycogen pool can be manipulated by fasting before slaughter which further affects the glycolytic rate and the pH decline post-mortem. A short fasting time of 4 h led to a delayed degradation of glycogen, slow decline in pH and a lower pHu in the LD compared with a fasting time of 26.5 h which resulted in a rapid breakdown of glycogen and pH decline early post-mortem and a high pHu. A long fasting time of 26.5 h was needed in order to affect glycogen stores at slaughter. Moreover, the results support earlier findings that proglycogen is degraded in favour of macroglycogen under anaerobic conditions post-mortem. The present data shows that feeding the animals in the morning before delivery if slaughtered the same day, results in low pH reduction rate and a low pHu compared to pigs fasted over night either on farm or at the abattoir. Aiming a higher pHu in LD it should be recommended not to feed the pigs in the morning at the day of slaughter. Finally, the detected sex differences on glycogen stores, glycogen degradation and pH develop-

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ment and the interactions with feeding regime need further studies in order to obtain a better understanding of the underlying mechanisms. Acknowledgements This project was funded by the Foundation for Research Levy of Agricultural Products, The Research Council of Norway and Felleskjøpet Fôrutvikling. The authors wish to thank Inge Lise Sørensen and Jens Askov Jensen for technical assistance and help with the analysis. The assistance and cooperation of Nortura Hed-Opp slaughterhouse staff, BioBank AS staff, Animalia, Norwegian Meat and Poultry Research Centre staff and technical staff is gratefully acknowledged. References Adamo, K. B., & Graham, T. E. (1998). Comparison of traditional measurements with macroglycogen and proglycogen analysis of muscle glycogen. Journal of Applied Physiology, 84, 908–913. Bee, G. (2002). Effect of available dietary carbohydrate on glycolytic potential and meat quality of swine muscles. Canadian Journal of Animal Science, 82, 311–320. Bertol, T. 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