Variation in daily feed intake and growth between Merino sheep lines selected for differences in resistance to nematodes.

Abstract

117 Variation in daily feed intake and growth between Merino sheep lines selected for differences in resistance to nematodes E.K. Doyle1, L.P. Kahn1, S.J. McClure2 and J.M. Lea1 1 2 School of Rural Science and Natural Resources, Animal Science, University of New England, Armidale NSW 2351 CSIRO Livestock Industries, Locked Bag 1, Armidale NSW 2350 edoyle2@metz.une.edu.au Summary This trial was designed to characterise daily feed intake and growth responses of animals selected for genetic difference in resistance to nematode infection when fed different quality diets, with and without an artificial infection with Haemonchus contortus. Fifty four Merino 10months old rams from the CSIRO flock with three divergent lines selected for either increased (IRH) or decreased (DRH) resistance to Haemonchus , or unselected (C) were stratified within selection line on the basis of liveweight measured following adjustment to experimental conditions and faecal egg counts from previous experimental challenge and individually housed during a two week noninfected period and nine weeks of trickle infection with McMaster strain of H. contortus. Throughout infection, there were no significant differences in daily feed intake and body weight gains between selection lines, but rams on the high quality diet had significantly higher (P<0.0001) feed intakes and liveweight gains than those fed the moderate quality diet. Average faecal egg counts per gram at week 9 of infection followed the expected differences between selection lines; the counts were 3,102 for IRH, 14,417 for DRH and 9,674 for C. It is assumed from the lower faecal egg counts that resistant animals were carrying significantly lower worm infections. The results suggest a difference in supply and/or partitioning of peptides and amino acids between selection lines, the IRH compared with DRH and C directing more of their protein supply into immune functions and less to production functions such as weight gain. Keywords: feed intake, resistance, genetic selection, nematode infection, Haemonchus contortus use. Resistance is the ability of a host to reduce the establishment, survival or reproductive rates of the parasite, whereas with resilience the host continues to maintain productivity despite infection (Gray 1995). There are a number of experimental flocks within Australia that have demonstrated the value of genetic selection for nematode resistance, one of which is the CSIRO Haemonchus selection flock (Woolaston et al. 1990). This flock consists of three divergent lines selected for either increased resistance to Haemonchus (IRH), decreased resistance to Haemonchus (DRH) and a randombred line (C). The IRH selection line has displayed lower faecal egg counts (FEC) (Woolaston et al. 1990, 1997) and reduced pasture contamination (Eady et al. 2003) compared with susceptible counterparts. There has been little work to elucidate the physiological and immunological responses that characterise the differing level of resistance against GI nematodes in the Haemonchus selection flock. There is evidence that selection for nematode resistance has correlated effects on the supply and/or partitioning of peptides and amino acids between the gut immune response and production functions, such as liveweight gain and wool growth. Doyle (1999) demonstrated that in the absence of GI parasites the IRH sheep have lower levels of wool production and liveweight gain than C sheep. More recently, Eady and Smith (2001) and Kahn et al. (2003) reported that IRH animals do not have greater levels of production compared to their C counterparts, despite having considerably lower burdens of GI nematodes as assessed from worm egg counts. The absence of a production advantage in the IRH line is unexpected because it could be presumed that nematodeinduced pathology and hence endogenous protein loss are reduced in these sheep, allowing utilization of additional protein for production. Therefore, this experiment was conducted to test two hypotheses: firstly, that animals from lines selected for differences in resistance to nematode infection would have different daily feed intakes when fed moderate and high quality diets in the absence and in the presence of Introduction Breeding for resistance to gastrointestinal (GI) nematodes is a longterm method of control that has been demonstrated to be an effective strategy to reduce susceptibility to infection and frequency of anthelmintic Recent Advances in Animal Nutrition in Australia, Volume 14 (2003) 118 Doyle et al. infection; and secondly that animals from these selection lines would have similar liveweight gains during infection when fed moderate or high quality diets. weeks where animals were maintained wormfree, followed by an infection period of nine weeks. Animals and housing Materials and methods Experimental design The sheep were kept in individual pens in an animal house. Seven weeks were allowed for the animals to adapt to experimental conditions and to complete a diet selection study (results of which are not reported here). Animals were fed experimental diets for three weeks prior to the experimental design described below. The experiment was a 3 x 2 factorial with three selection lines selected for genetic difference in resistance to nematode infection and two diets, one a high quality (high metabolizable protein and metabolizable energy) and the other of moderate quality (moderate metabolizable protein and metabolizable energy). They were stratified within selection line on the basis of liveweight measured following adjustment to experimental conditions and faecal egg counts (FEC) from a previous experimental challenge, and randomly allocated to diets and position of pen. The experiment was conducted over two periods, firstly a period of two The study used fiftyfour Merino weaner rams selected randomly from the CSIRO Haemonchus IRH (n = 19), DRH (n = 15) and C (n = 20) selection lines (Woolaston et al. 1990). They were approximately 10 months of age at the beginning of the experiment with a mean � SD liveweight of 26.1 � 2.85 kg. They had previously been artificially infected with 10 000 L3 H. contortus at approximately six months of age and subsequently exposed to natural field infection. This infection history was important to ensure animals had prior opportunity to acquire immunity to nematode infection and reflect genetic merit. Upon entering the animal house each animal was drenched with Scanda� (8mg/kg levamisole hydrochloride and 4.5 mg/kg oxfendazole, Schering Plough Animal Health Ltd) and Ivomec� (0.2 mg/kg ivermectin, Merial), to remove existing worm burdens, and were given an intermuscular injection of vitamin B12 (1 mL/animal, hydroxocobalamin and cyanocobalamin, Novartis Animal Health). A faecal egg count was taken 5 days after drenching to ensure all animals had zero counts. Table 1 Ingredients1 and chemical analysis of experimental diets. High quality Ingredients (% per kg fresh matter) Cottonseed hulls Barley Lucerne chaff Molasses Cottonseed meal Dry matter (% as fed) Organic matter (% as fed) DigestibilityA (% DM) Ether extract (% DM) Crude protein (% DM) Calculated MP (g/kg DM) Calculated ME C (MJ/kg DM) Sulphur (g/kg DM) Phosphorus (g/kg DM) Iron (ppm) Copper (ppm) 1 Mod quality 1 Mod quality 2 39.0 16.0 15.0 8.0 22.0 90.8 86.8 62.7 3.0 16.8 90.0 9.2 2.1 3.3 147.2 7.1 54.0 23.0 10.0 10.0 3.0 92.3 88.9 58.3 2.0 9.7 43.0 8.3 1.7 2.2 154.5 5.4 77.0 11.5 5.0 5.0 1.5 89.9 89.2 46.1 1.6 7.5 30.4 6.3 2.0 2.4 160.3 6.0 B Major minerals and trace elements were added to make diets similar in concentrations. Averaged across diets these per kg dry matter were 3.25 g Ca, 2.6 g Mg, 12.8 g K, 1.213 g Na, 42 mg Zn, 48 mg Mn and 2 mg Mo. Feed was analysed for mineral concentrations using the Vista MPX radially viewed, simultaneous Inductively Coupled Plasma Optical Emission Spectrometer (ICP_OES) A Based on pepsin cellulase wet chemistry B Calculated from equations of Freer et al. (1997) C Calculated from algorithm supplied by FeedTest, Victorian Department of Primary Industries, Hamilton Vic 3300 . Performance of Merinos differing in resistance to nematodes 119 Feed The composition and analysis of the experimental diets are given in Table 1. Diets were formulated to be non limiting and balanced for minerals, and were pelleted to reduce selectivity of each feed component. Diet formulation was not constrained to make diets isoenergetic while differing in metabolizable protein (MP) content. Donaldson et al. (1998) have shown that it is the level of MP, not metabolizable energy (ME), which has the most influence on host resistance to nematode infection. Animals were offered quantities of fresh food once daily that were 10% greater than ad libitum intakes on the previous day. A subsample of food refusals from each animal was taken weekly for later analysis of remaining contents. The high quality diet (H) was fed throughout the experimental period. Two moderate quality diets were used. Moderate quality diet 1 (M1) was fed in the non infection period only and then changed to the moderate quality diet 2 (M2) at the start of the infection; this change was instigated by unexpectedly high growth rates on M1, and was to ensure animals would be able to differentiate between the moderate and high quality diets for the diet selection study. salt solution. The higher specific gravity (1.4) of potassium iodine was required to enhance flotation and therefore detection of eggs from animals consuming the pelleted diets rich in cottonseed hulls. Statistical analysis All statistical analyses were performed using the SAS computer program (SAS Institute Inc 19992001). Generalised Linear Models (GLM) were used to analyse the significance of diet quality (H and M), selection line (IRH, DRH and C) and the interaction (diet x line) between these effects. The models used repeated measures analysis of variance for daily feed intake, body weight gain and faecal egg counts. Initial liveweight was not a significant covariate for subsequent liveweight gain measures. Change in liveweight during the uninfected period was not of primary interest and, because this was only 2 weeks long, statistical analysis was not considered reliable. Analysis was performed separately for each period (with and without infection) and also because of the change from M1 to M2 diet. Least squares means � SE are presented for daily feed intake and weight gain. Faecal egg counts were cube root transformed to normalize data prior to analysis and are presented as backtransformed means. Infection All animals remained parasite free for the first period of the trial. In the second period all animals were infected with McMaster strain H. contortus L3. An initial infective dose of 150 L3/kg liveweight was administered orally at the beginning of the infection period and followed by a trickle infection of 250 L3 three times per week on Monday, Wednesday and Friday (average total dose � SD over the infection period of 12 570 � 565 L3/sheep). Results Daily feed intake During the 2 week noninfection period, selection line significantly (P = 0.03) affected daily feed intake. Weaner rams from the IRH line had greater feed intake than the C line but there was no difference in intake between C and DRH (Table 2). Diet had no affect on daily feed intake and there was no significant interaction between the effects of selection line and diet. The effect of the independent variables selection line and diet on the daily feed intake (g DM/day) of the weaner rams with an artificial infection of Haemonchus contortus is displayed in Figure 1. During the infection period the daily feed intake did not differ between selection lines despite a large and significant (P<0.0001) difference in faecal egg counts. Conversely, diet did significantly (P<0.0001) Animal measurements Animals were weighed in the morning prior to feeding at the start of the experiment and then weekly during both periods that followed. A faecal egg count per animal was measured at days 0 and 21 after the initial dose of infective larvae and weekly thereafter. A modified McMaster technique was used to count worm eggs, but potassium iodine was used rather than sodium chloride Table 2 Average daily feed intake (g DM/day) of weaner rams, from lines that had been selected for either increased resistance to Haemonchus (IRH), decreased resistance to Haemonchus (DRH) and random_bred control (C), without nematode infection. Selection line IRH DRH C Least squares means 1613 1508 a ab Standard error 51.6 58.2 50.3 1415b Least square means with different subscripts differ significantly (P<0.05) 120 Doyle et al. Feed Intake (g DM/day) affect daily feed intake of infected animals (Figure 2). Interactions between the effects of selection line and diet were not statistically significant during the infection period. (a) 1800 1700 1600 1500 1400 1300 1200 Feed Intake (g DM/day) 1700 1600 1500 1400 1300 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Weeks of i nfection Figure 2 Daily feed intake (g DM/day � SE) of weaner rams artifically infected with Haemonchus contortus and fed different diets of high ( and moderate ( ) quality. ) (b) 1700 W ee ks of infe ction Feed Intake (g DM/day) 1600 1500 1400 1300 1200 1100 1 2 3 4 5 6 7 8 9 The effect of selection line on liveweight change was not significant during the infection period. There was also no significant interaction between the effects of selection line and diet for liveweight gain. Faecal egg count Faecal egg counts followed the expected differences between selection lines; the average FEC per gram of faeces at week 9 of infection was 3,102 for IRH, 14,417 for DRH and 9,674 for C rams. Analysis showed a significant (P<0.0001) selection line effect throughout the infection period. There was no effect of diet on FEC throughout the infection, nor was there a significant interaction between the effects of selection line and diet. Weeks of infection Figure 1 Daily feed intake (g DM/day � SE) of IRH ), DRH ( ) and C ( ) weaner ( rams differing in their genetic resistance to Haemonchus contortus, with an artificial infection of H. contortus and fed high quality (a) and moderate quality (b) diets. Discussion Daily feed intake The first objective of this study was to investigate whether animals from lines selected for differences in resistance to nematode infection would have different daily feed intakes when fed two different quality diets in the absence and presence of infection. Results found the IRH line had significantly higher daily feed intakes during the noninfected period, compared to the C line, with the DRH line being intermediate. However, when Liveweight change Animals on both the H and M quality diets gained liveweight throughout the infection period (Table 3). There was a significant difference (P <0.0001) in liveweight gain between diets during the infection period. On average weaner rams fed H gained 11.8 kg during the 9 week infection period, while animals fed M2 gained an average of 5.8 kg. Table 3 Liveweight gain (kg) of weaner rams, from lines that had been selected for either increased resistance to Haemonchus (IRH), decreased resistance (DRH) and random_bred control (C) calculated over the 9 week period of artificial infection with H. contortus. Selection line IRH DRH C Least squares mean 11.9 11.5 12.0 5.7 7.0 4.9 Standard error 0.71 0.75 0.67 0.67 0.81 0.68 Quality of diet High Moderate IRH DRH C Performance of Merinos differing in resistance to nematodes 121 the same animals were artificially challenged with H. contortus the selection line effect on intake was not apparent. The start of the infection regime coincided with the introduction of the M2 diet for reasons discussed earlier. Feed intake for all selection lines declined uniformly with the introduction of M2 and the start of infection. The decrease in feed intake on the M2 diet occurred within one week of its introduction, suggesting that the change from M1 to M2 diets rather than the beginning of the H. contortus infection was the underpinning factor for the intake decline. Further experimentation is required to determine if the significant difference in feed intake between selection lines during the noninfected period would have remained apparent during the infection period if M1 had continued to be fed. Diet significantly influenced the daily feed intake of each selection line during the infection period with higher intakes recorded for the H diet. The effect of diet on feed intake of animals infected with H. contortus has previously been demonstrated by Datta et al. (1998), who found that voluntary feed intake of infected lambs increased with increasing dietary crude protein. The nonsignificant difference between the diets for feed intake in the noninfection period may have been because differences between dietary levels of MP and ME were not sufficient to allow expression of feed intake differences. DRH line showed similar liveweight gains as the IRH line while carrying an infection almost fivefold that of the IRH line. Clearly these susceptible animals are displaying strong resilience to Haemonchus challenge. In further support for this working hypothesis, weight gains of animals eating the M2 diet varied between selection lines, although differences were not significant. The DRH line gained 1.3 kg more than the IRH line over the infection period, despite the two selection lines having similar daily feed intakes. Analysis showed a trend (P = 0.091) of dry matter intake adjusted for body weight (DMI/BW), showing the IRH line did eat a higher proportion of dry matter per unit body weight than the DRH. This may suggest that with lower quality diets the metabolic cost of resistance is greater than the metabolic cost of infection (WalkdenBrown and Eady 2003). Liveweight gain during the infection was far greater than predicted (Freer et al. 1997). The reasons are currently unknown, but the cottonseed hulls used in the experimental diets may play a role in the greater weight gains. The H diet supported larger weight gains (187 g/d) during infection compared to the M2 diet (93 g/d). The importance of diet on resilience to infection has previously been shown in European breeds of sheep also infected with H. contortus, where animals fed high protein diets gained more weight than those fed low protein diets (Abbott et al. 1985a; Wallace et al. 1995, 1996). Liveweight change Animals from lines selected for differences in resistance to nematode infection had similar liveweight gains during the infection period on both high and moderate quality diets despite a significant difference in FEC. One would expect the IRH line to have had higher weight gains than the animals from the DRH and C lines, as these animals had considerably lower FEC. However this result validates previous work with pregnant and lactating ewes (Kahn et al. 2003) and weaners (Eady and Smith 2001) from the same Haemonchus selection lines; weight changes of IRH and C lines were similar whether uninfected or infected. A possible explanation for this recurring observation is that divergent selection for resistance has produced changes in supply and/or partitioning of peptides and amino acids between the gut mucosal immune response and production functions, such as weight gain and wool growth, in each selection line. The basis of these changes may originate in the selection process used to identify resistant animals, which have been selected on their ability to more effectively prevent establishment and/or development of nematode infection. The IRH animals may continue to redirect their protein supply away from body growth into immune functions to prevent larval establishment and maintain resistance, even in the absence of infection. It is possible that resistant animals may not be able to effectively down regulate their mucosal immune response, resulting in excessive activity. In contrast, the Faecal egg count Woolaston et al. (1990) have observed the distinct selection line affect on FEC. The nonsignificant effect of diet quality on FEC supports earlier work by Abbott et al. (1985a,b) and Wallace et al. (1996) who found that supplementation with protein did not improve the development of immunity in breeds of sheep (e.g. Scottish Blackface) which are relatively resistant to nematode infection. However, Abbott et al. (1985a,b) and Wallace et al. ( 1995) also found that breeds susceptible to nematode infection benefited from dietary protein supplementation. In this experiment there was a trend for animals from the DRH and C lines to have lower FEC on the H diet. One reason dietary effects on FEC may not have been greater, is the quality of the M2 diet was sufficiently high to allow expression of resistance. In conclusion we have found firstly that in the absence of infection the IRH line has significantly higher daily feed intakes compared to the C selection line. Secondly, there were no differences in daily feed intake or weight gain between selection lines during infection, despite a significant difference in faecal egg count. 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