Immunization of entire males against gonadotrophin releasing factor (GnRF) is an effective strategy to eliminate boar taint and a welfare friendly alternative to physical castration [1
]. It involves two immunizations with an incomplete analogue of GnRF conjugated to a carrier protein in a low reactogenic-adjuvant system [2
]. Following the second immunization the pig has an increased feed intake and growth rate but there is also an increase in backfat and whole body fat deposition compared to entire males [2
]. Management strategies to limit the increase in feed intake and fat deposition and promote lean deposition are required for markets where producers are penalized for high backfats.
A way to reduce the increase in feed intake and increase in fat in immunocastrated (IC) male pigs is to restrict feed intake as restrictively fed IC male pigs have a reduced backfat [9
] and increased carcass leanness [10
]. However, restricting feed intake by restricting the amount of feed in a group-housed environment has welfare issues in terms of increased aggression [10
]. If a feeding strategy can be identified that reduces feed intake of IC males to similar to that of entire males, then this could be a sound management strategy to limit the increase in feed intake and fat deposition associated with the production of IC male pigs.
The aim of this experiment was to use feed ingredients such as Lupinus albus
(albus lupins) or a combination of calcium chloride and sodium tri-phosphate to suppress the voluntary feed intake of IC male pigs when fed ad libitum. The possible effect of these ingredients on objective meat quality was also explored to ensure that the meat quality was not negatively impacted. Albus lupins have been found to reduce feed intake in several pig experiments [11
]. Dunshea et al. [11
] suggested that the most likely mechanism by which albus lupins affect feed intake is by delayed transit in the stomach and small intestine. This delayed transit then feedbacks on satiety signals. A combination of calcium chloride and sodium tri-phosphate (mineral salts) has reduced feed intake in the range of 6% to 15% [13
]. It suppresses the pig’s appetite by changing the acid to base ratio by increasing the plasma chloride level and subsequently creating a HCO3−
The hypotheses tested in this study were (1) pigs immunized against GnRF which are fed either a diet containing albus lupins or mineral salts will have a reduced feed intake with no effect on growth rate compared to pigs receiving a standard finisher diet; (2) pigs fed either albus lupins or mineral salts will deposit less fat compared to pigs receiving a standard finisher diet; (3) pigs immunized against GnRF and fed either a diet containing albus lupins or mineral salts will have a similar backfat compared to entire males receiving a standard finisher diet and; (4) feeding pigs a diet containing albus lupins or mineral salts will have no effect on objective meat quality compared to a standard finisher diet.
2. Materials and Methods
The experimental protocol used was approved by the Department of Agriculture and Food Western Australia’s Animal Research Committee and by the Animal Ethics Committee (activity number 1-15-02). The animals were handled according to the Australian code of practice for the care and use of animals for scientific purposes [15
]. A total of 294 Large White × Landrace × Duroc entire male and immunocastrated male pigs were used in this experiment. The experiment was a 2 × 3 factorial with the main treatments being: (i) sex and lysine concentration (sex; entire males fed a diet with 0.64 g standardized ileal digestible (SID) lysine/MJ DE (mega joule digestible energy) for four weeks (entire males) or IC males fed a diet with 0.64 g SID lysine/MJ DE for two weeks followed by 0.50 g SID lysine/MJ DE for two weeks (IC male)); and (ii) feed ingredient (diet; control, 3% calcium chloride + 1.6% sodium tripolyphosphate, or 300 g/kg albus lupins).
2.1. Allocation and Housing
Pigs were sourced from a high health status commercial herd at 34.9 ± 3.98 kg (mean ± SD) liveweight (LW). Upon arrival pigs were individually identified with ear tags, weighed and stratified on their LW. The allocated pigs received a priming dose of anti-gonadotrophin releasing factor immunological product (Improvac®, Zoetis Australia, Rhodes, Australia) on d −28 (where d 0 is when all pigs received the second dose of the anti-gonadotrophin releasing factor vaccine). Pigs were group housed (n = 7) in a naturally ventilated grower shed. They had ad libitum access to water, and a commercial feed via a single spaced feeder.
2.2. Diets and Feeding Regime
On d 0 all pigs received the experimental diet and the second dose of the anti-gonadotrophin releasing factor vaccine was given to the allocated pigs who had received the priming dose on d −28. The entire males did not receive a placebo injection. The experimental diets were formulated to the same nutrient specifications (14 MJ DE and 0.64 g SID lysine/MJ DE (high) or 0.50 g SID lysine/MJ DE (low)). The diets were formulated so that the IC male pigs were fed as entire males for 2 weeks (from d 0; high) and then the lysine level in the diet was reduced for the remaining 2 weeks (low; based on recommendations from Moore et al. [7
]). The entire male pigs continued to receive the diet adequate for an entire male pig (high). The composition of the experimental diets is given in Table 1
. The diets were also analyzed for quantitative amino acid composition (Australian Proteome Analysis Facility, Sydney, Australia) and the results are presented in Table 2
2.3. Growth Performance
Pigs were weighed weekly and feed refusals determined on d 0, 7, 14, 21 and 28 to measure average daily gain and voluntary feed intake. The feed conversion ratio was calculated on a weekly basis from when the feeding of the experimental diets commenced.
2.4. Dual-Energy X-ray Absorptiometry Analysis
Twelve pigs per treatment (3 pigs/pen randomly selected from 4 replicate pens, so 72 in total (12 pigs × 6 treatments)) were scanned on d −1, 13 and 27 using dual-energy x-ray absorptiometry (DXA). The pigs were removed from feed and fasted for approximately 16 h before scanning. Immediately before scanning the pigs were weighed and then transferred to the DXA facility. They were injected intramuscularly with Stresnil®
(azaperone 40 mg/mL, Stresnil Neuroleptic Injection for Pigs, Ausrichter Pty Ltd., Newtown, Australia) at 2 mL/10 kg LW. When sufficiently sedated the pigs were transferred to the DXA machine (Norland XR46 Densitometer Machine, Norland Products Inc., Cranbury, NJ, USA) [16
]. The pigs were scanned in ventral-recumbency, with hind legs extended and forelegs positioned caudally. Whole body mode was used to scan and the scan was subsequently analyzed using whole body analysis. Measurements made by DXA included lean tissue mass, fat tissue mass and bone mineral content. After scanning the pigs were placed in a recovery room until they were able to stand and were then returned to their pens. The pigs were given their respective diets on return to their individual pens.
2.5. Slaughter Procedure
Four weeks after the diets were introduced pigs were individually tattooed, removed from feed overnight and transported to a commercial abattoir (approximately 90 min transport time). The pigs were stunned using a carbon dioxide, dip-lift stunner set at 85% CO2 for 1.8 min (Butina, Holbæk, Denmark). Exsanguination, scalding, dehairing and evisceration were performed using standard commercial procedures. Hot carcass weight (HCW, AUSMEAT Trim 13; head off, fore trotters off, hind trotters on; AUS-MEAT Ltd., South Brisbane, Australia) and P2 backfat depth, 65 mm from the dorsal midline at the point of the last rib (PorkScan Pty Ltd., Canberra, Australia) were measured approximately 35 min after exsanguination, prior to chiller entry (2 °C, airspeed 4 m/s).
2.6. Blood Analysis
Blood samples (20 mL in lithium heparin tubes) were collected on d 0, 7, 14, 21 and 28 from the same pigs that were selected for DXA scanning. The blood samples were centrifuged at 2000× g for 15 min to recover plasma and were stored at −20 °C until analysed. Plasma urea nitrogen (PUN) was quantified using a commercial kit (Beckman Coulter/Olympus Reagent Kit Cat. No. OSR6134 Lot #6042). Plasma urea (mmol/L) was converted to PUN (mg/dL) by dividing by 0.357. Calcium was quantified using a commercial kit (Beckman Coulter/Olympus Reagent Kit Cat. No. OSR60117 Lot #6564). Phosphorus was quantified using a commercial kit (Beckman Coulter/Olympus Reagent Kit Cat. No. OSR6122 Lot #5797). Glucose was quantified using a commercial kit (Beckman Coulter/Olympus Reagent Kit Cat. No. OSR6121 Lot #5512). The assays for PUN, calcium, phosphorus and glucose were performed on an automated analyzer according to the manufacturer’s instructions (Olympus AU400; Olympus UK Ltd., Hertfordshire, UK). Plasma leptin was determined using a commercial ELISA kit (Cusabio Pig Leptin ELISA (CSB-E06815p, Jomar Life Research Pty Ltd., VIC, Australia). Sodium and chloride were measured by indirect ion selective electrodes and the assays were performed on an automated analyzer (AU680, Beckman Coulter, Sydney, Australia). Total carbon dioxide was determined by reaction with phophoenolpyruvate carboxylase reagent and the assay was performed on an automated analyzer (AU680, Beckman Coulter, Sydney, Australia).
2.7. Objective Meat Quality
Twenty-one pigs per treatment (3 pigs/pen that were DXA’d (from 4 replicate pens) and an additional 3 pigs/pen which were randomly selected from the remaining 3 replicate pens) were used to assess meat quality. pH and temperature decline in the Longissimus thoracis
(LT) was measured at 45 min post-exsanguination using a portable pH/temperature meter (Cyberscan pH 300, Eutech Instruments, Singapore) fitted with a polypropylene spear-type gel electrode (Ionode IJ44, Ionode Pty Ltd., Brisbane, Australia) and a temperature probe. The pH meter was calibrated on two standards (pH 4.01 and 7.0) as per the manufacturer’s instructions. At 24 h post-slaughter a section of the LT muscle was removed from the left hand side of the carcass between the 12th and 13th rib. For determination of pH and temperature a 2 cm steak was cut from the appropriate sample and measured using the pH/temperature meter as previously described. Drip loss was measured using a modification of the method described by Rasmussen and Andersson [17
]. The muscle was cut to a 50 g cube then wrapped in netting and suspended in a sealed plastic container. The samples were stored for 24 h at 4 °C. The sample was then removed and gently patted dry to remove excess moisture before being re-weighed. Colour (L*, a* and b*) was measured with a Minolta Chromameter CR-400 (Minolta, Osaka, Japan), using D65 illumination, a 2° standard observer, and an 8-mm aperture in the measuring head, standardized to a white tile after a bloom time of 30 min (determined from the freshly cut 2 cm steak). An 80 ± 10 g (approximately 5 cm × 2 cm × 2 cm) sample was cut from the loin samples to measure cooking loss and shear force [18
]. The samples were frozen in individual bags. The bagged frozen samples were then suspended from a metal rack and placed in a water bath which had been pre-heated to 70 °C. The samples were cooked at 70 °C until an internal temperature of 70 °C was reached (approximately 30 min). After removal from the water bath, the samples were allowed to cool in iced water for 30 min, patted dry to remove excess moisture, and re-weighed before being refrigerated at 4 °C overnight. Cooking loss percentage for each sample was determined by dividing the difference in the raw and cooked weights by the weight of the raw pork sample. The cooked sample was then cut into five cross-section samples (1 cm2
) parallel to the muscle fibres. Warner Bratzler shear force was measured using a Warner Bratzler shear blade fitted to a Lloyd Texture Analyzer (TA-2, Lloyd Instruments Ltd., Bognor Regis, UK).
2.8. Statistical Analysis
General analysis of variance was performed with the GENSTAT 16 program (VSN International Ltd., Hemel Hempstead, UK) to analyze the main effects of sex and lysine concentration and feed ingredient on growth performance, carcass quality, body composition, physiological measures and objective pork quality measures. For growth performance, and carcass data, the pen was the experimental unit. For the DXA, physiological measures and objective pork quality measures, pig was the experimental unit. Repeated measures analysis of variance was used to analyze the blood measures. Batch was used as a block in the analysis. A level of probability of less than 0.05 was used to determine statistical difference between the means. A level of probability of less than 0.1 but greater than 0.05 was determined to be a trend. Fisher’s-protected least significant differences were used to determine differences among treatments.
The lysine concentrations in the diets of the entire males and the IC males were determined based on findings by Moore et al. [7
]. They found that IC males show a response to dietary SID lysine similar to that of entire males for two weeks after the second immunization against GnRF. After this, IC males have a lower requirement for SID lysine than entire males. In the current experiment there was no reduction in the growth rate and feed intake of the IC males fed the control diet when the lysine concentration was reduced in the final two-week period. This indicates that the reduction in feed intake is more likely to be attributed to either the albus lupins or mineral salts and so the results have been discussed from this perspective.
The hypothesis that pigs immunized against GnRF and which were fed either a diet containing albus lupins or mineral salts would have a reduced feed intake with no effect on growth rate compared to pigs receiving a standard finisher diet was partially supported. Immunocastrated male pigs fed the mineral salt diet ate less feed than those fed the control diet with no effect on growth rate. However, IC male pigs fed the albus lupin diet had both a reduced feed intake and growth rate compared to those fed the standard finisher diet.
The results for the mineral salt diet are in contrast to Yen et al. [13
] and Pluske et al. [14
] who found pigs fed a mineral salt diet had both a lower daily feed intake and weight gain compared to those on the basal diet. However, Yen et al. [13
] fed 4% CaCl2
O and 2.22% Na5
compared to the concentration of 3% CaCl2
O and 1.6% Na5
in this experiment. Pluske et al. [14
] used 4% CaCl2
and 2.2% Na5
for one week before halving the concentration for the remaining two weeks due to acceptance issues of the diet in the first week. The differences in concentration levels between studies may help to explain the observed differences in performance. In addition, the acceptance issues in Pluske et al. [14
] may be because calcium chloride was used rather than calcium chloride dihydrate which increased the effective level of calcium chloride to 94% compared to 77% calcium chloride both in the current experiment and in Yen et al. [13
The most likely explanation by which CaCl2
reduced feed intake is through the process of metabolic acidosis. Yen et al. [13
] found that the acidosis is caused by an increase in the plasma chloride level which sets off a number of events resulting in a reduction in the buffering capacity of the HCO3−
. This then leads to a low blood pH and a reduction in total CO2
. We also found an increased plasma chloride concentration and a reduction in CO2
. However, we did not measure blood pH or HCO3−
. Although there was an increase in the dietary concentration of calcium this was not reflected in the plasma calcium concentrations because the calcium is excreted as Ca3
in the faeces [13
The albus lupins were originally included in the diet at 30% based on findings from Dunshea et al. [11
] and Van Nevel et al. [12
]. These researchers observed reductions in feed intake in the finishing period of between 12% and 27% [11
]. We were trying to decrease the feed intake of the IC males to similar intake levels of entire males, a reduction of approximately 15% which corresponded with the previous findings of reductions in intake. However, in the current experiment from d 0 to 7 feed intake of the pigs receiving the albus lupin diet was nearly halved compared to the control diet. It is suggested that the observed differences in intake between the experiments may be because the pigs in the ad libitum fed experiment in Dunshea et al. [11
] and in van Nevel et al. [12
] were acclimatized to the albus lupin diet before the experimental period. There was no acclimatization to diet in the present study.
When the daily gain, feed intake and feed conversion ratio data were examined for the d 15–28 period only and the pigs were fed a diet with 20% albus lupins, the IC male pigs on the albus lupin diet had a similar daily gain (1.05 vs. 1.09 kg/d), feed intake (3.03 vs. 3.05 kg/d) and feed conversion ratio (2.90 vs. 2.81 kg/kg) compared to entire males fed the control diet. Although there were differences in the lysine concentrations between the diets fed to IC males and entire males, the lysine concentrations were based on findings from Moore et al. [7
] and were formulated to meet the requirements of the pigs at the weights and sex and were unlikely to be the factor affecting the daily gain and feed conversion. Therefore, there is potential for albus lupins to reduce the feed intake of IC males when included in the diet at 20%, however, growth rate was also reduced to similar to that of entire males.
The hypothesis that pigs fed either albus lupins or mineral salts will deposit less fat compared to pigs receiving a standard finisher (control) diet was partially supported. Irrespective of sex, pigs fed albus lupins deposited less fat compared to those on both the control and mineral salt diet. There was no difference in fat deposition between pigs fed the control or mineral salt diet. As far as we are aware there is no previous research which has investigated using these in-feed ingredients to reduce feed intake with the main objective being to reduce fat deposition.
Given that there was a reduction in feed intake in pigs receiving the mineral salt diet it was anticipated that there would be a corresponding reduction in fat deposition. However, even though there was a reduction in feed intake of 9.2% for the IC males on the mineral salt diet compared to the control diet perhaps this was insufficient to promote a decrease in fat deposition. The IC male pigs fed the mineral salt diet still consumed 9.3% more feed than the entire males fed the control diet. When diets of IC male pigs have been restricted previously by restricting the amount of feed reductions in backfat have been observed when the feed intakes were between 15% and 22% lower than pigs fed ad libitum [9
The hypothesis that pigs immunized against GnRF and fed either a diet containing albus lupins or mineral salts would have a similar backfat compared to entire males receiving a standard finisher diet was partially supported. Immunocastrated male pigs fed the albus lupin diet had a similar backfat compared to entire males receiving the standard finisher diet. However, IC males fed the mineral salt diet were 1.36 mm fatter than entire males receiving the control diet and had a similar backfat to IC males receiving the control diet. Therefore, albus lupins show potential in reducing the increase in backfat associated with immunocastration. Further investigation should be undertaken to determine the effect of including a constant 20% albus lupins in the diet of IC males for either 28 or 14 days pre-slaughter. Van Nevel et al. [12
] also found that including albus lupins in diets at 30% reduced backfat thickness with a tendency for the percentage of lean content to increase. The reduction in backfat thickness and increase in lean was attributed to the slower growth rates [12
Plasma leptin was lower in IC males to entire males from d 7 after the second immunization against GnRF. This is in contrast with previous studies which found that plasma leptin concentrations increased in IC males compared to entire males [4
]. Leptin is positively correlated with the amount of fat in the body [20
]. When leptin is given endogenously there is a reduction in feed intake [22
]. There is an increase in feed intake following the second immunization against GnRF and it is thought that plasma leptin increases to try to decrease feed intake to combat the increase in body fat [4
]. Therefore, it was expected that plasma leptin would increase as both feed intake and fat deposition increased in IC males in the current experiment and it is not known why this was not the case. The leptin concentrations were also higher in the current experiment compared to McCauley et al. [4
] and Batorek et al. [10
]. This can be attributed to differences in the assay type (multi-species radioimmunoassay method versus a pig specific competitive ELISA method) and leptin specificity of the respective methods [23
The hypothesis that feeding pigs a diet containing albus lupins or mineral salts will have no effect on objective meat quality compared to a standard finisher diet was partly supported. There was no difference in objective meat quality between any diets with the exception of pH45 min
which was higher in meat from pigs fed the albus lupin diet and the mineral salt diet compared to the control. When including alternative ingredients in pig diets it is important to ensure that there is no adverse impact on the meat quality of the diets. There does not appear to be any other research investigating the effect of including mineral salts in the diet on meat quality. Kim et al. [24
] investigated including Lupinus angustifolius
in pig diets at 350 g/kg and found no effect on meat quality. Although there does not appear to be any other work investigating the effect of albus lupins on meat quality in pigs, there has been some work in young bulls. When young bulls were fed diets containing either 20% albus lupins or 16.5% soybean there were no differences between diets for meat quality [25
The IC males had an increased drip loss which is associated with the lower ultimate pH and higher L value (lighter) observed compared to entire males. This concurs with a meta-analysis by Batorek et al. [26
] who found that IC males tend to have a lower ultimate pH, a higher L* value (are lighter) and an increased drip loss compared to entire males. Immunocastrated male pigs have reduced aggressive behaviour and physical activity compared to entire males and this is likely associated with the lower ultimate pH [3
]. However, the meta-analysis by Batorek et al. [26
] also found that IC males also have a lower shear force and no difference in the degree of yellowness compared to entire males and this was not observed in the present study.