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* Pepscan Systems B.V., 8219 PH Lelystad, The Netherlands;
and
Isotope Research Lab, Sichuan Agriculture University, Ya’an, Sichuan 625014, P.R. China; and
and
ID-Lelystad, Institute for Animal Science and Health, 8219 PH Lelystad, The Netherlands
2 Correspondence:
Edelhertweg 15, 8219 PH Lelystad, The Netherlands (phone: 31.320.237212; fax: 31.320.238120; E-mail:
j.a.turkstra{at}id.wag-ur.nl.).
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: GnRH • Growth Rate • Immunization • Pigs • Testes
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Immunocastration (i.e., active immunization against GnRH) may offer an alternative for surgical castration (Meloen et al., 1994; Oonk et al., 1998; Hennessy et al., 1997). In boars that are immunized against GnRH, the concentrations of androstenone and skatole (both contributing to boar taint; Matthews et al., 2000) are at the level of surgical castrates, resulting in taint-free pork (Hennessy et al., 1997).
Immunocastration preferably is performed in the finisher phase, in order to utilize maximally the boar-type growth during the period before the onset of immunocastration (Bonneau et al., 1994). However, immunocastration shortly before slaughter does not allow discrimination between immunocastrated boars and intact boars by simply estimating the testis size. In our study, therefore, we applied immunization at 9 and 17 wk of age, which has shown to meet the criteria of visible reduced testis size (Oonk et al., 1995). In previous experiments, we observed a considerable variation between animals in their response to the immunizations. We hypothesized that a more boarlike performance can be expected in GnRH-immunized boars with a "late" response (as determined by substantial LH and testosterone levels still being present at the time of second immunization) than in GnRH-immunized boars with an "early" response or in surgical castrates.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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The experimental protocol describing the management, surgical procedures, and animal care was reviewed and approved by the Dutch Committee on Animal Care and Ethics. Forty-eight piglets from 16 litters of Dutch commercial crossbred pigs (Dutch Landrace x Finnish Landrace) x Large White were selected in the first week after birth. From each litter, three male piglets were chosen. Of these, one was surgically castrated between 8 and 18 d after birth, another served as an intact control, and one was immunized at 9 and 17 wk of age. The three piglets from each litter were randomly assigned to the treatment groups.
Two days before the start of the experiment, the three littermates were allocated to the same pen, with three pigs per pen. The pens consisted of two sections: one section for individual feeding, and a group section with a half-slatted floor for lying and dunging, and in which section additional water was supplied. The animals were fed individually two times a day. Feed intake was restricted to 2.8 times MEm (418 kJ ME/W0.75) at the start of the experiment and was gradually increased to 3.2 times MEm. From the start of the experiment, at 23 kg BW, until the pigs weighed 45 kg, a starter diet was fed, and from 45 kg BW to slaughter, a grower diet was supplied. The feed was offered together with water at a ratio of 1:2.5 in the trough, whereas the pigs had free access to water in the group section. The diets were composed of feedstuffs commonly used in The Netherlands (mainly barley, soybean meal, and tapioca). They were formulated to contain 110% of the current Dutch recommendations of amino acids for slaughter pigs in order to allow the boars to fully utilize their growth potential (CVB, 1997). During manufacture of the diets, triplicate samples were taken and analyzed according to the methods described by AOAC (1984).
Experimental Design and Treatments
One intact male piglet out of each triplet was immunized at 9 wk of age (d 0) and at 17 wk of age (8 wk post vaccination, 8 wk) by i.m. injection at the left and right side of the neck region, respectively. The vaccine consisted of 1 mg of synthetic GnRH-tandem peptide in 2 mL of Freund’s complete adjuvant emulsion (Oonk et al., 1998). For the second immunization, Freund’s incomplete adjuvant was used. The remaining intact male piglet of each triplet was not injected. The immunized pigs were retrospectively divided into two categories, based on their LH and testosterone levels at the time of booster immunization (i.e., at 8 wk after the first immunization). Immunized pigs were defined as "early" responders (E-IM) when both LH and testosterone concentrations at 8 wk were low. Testosterone concentrations should be similar to those of surgical castrates (< 0.40 pmol/mL), and LH should be less than 0.80 ng/mL. Immunized pigs that had testosterone concentrations at 8 wk exceeding the level of surgical castrates were defined as "late" responders (L-IM). Luteinizing hormone levels of the L-IM pigs were variable, and ranged between 0.63 and 1.95 ng/mL. The E-IM and the L-IM group consisted of eight and seven pigs, respectively. One triplet group was excluded from the analysis of the experiment results, because the immunized pig did not respond to the immunizations. In contrast to the other immunized pigs, this pig exhibited substantial serum LH and testosterone levels throughout the experiment and the weight of its testes was similar to that found in intact boars.
Measurements
Animals were weighed weekly in order to determine the amount of feed that should be supplied. Blood samples were taken at d 0, 8 wk, 12 wk, and 1 d before slaughter. Blood samples were taken via puncture of the vena jugularis and kept overnight at 4°C. The next day, serum was obtained by centrifugation (2,000 x g, 15 min). Serum samples were stored at -20°C until assayed. Testis size was determined by measuring testis length using vernier calipers at each day of blood sampling; mean values of the two testes were recorded. At 90 kg BW, and at 1 d before slaughter, ultrasonic backfat measurements were performed. Backfat thickness was measured at four positions on the left side and at four positions on the right side of the median. Measurements were performed according to the method described by Walstra (1987). Pigs were killed 1 wk after they weighed at least 102 kg. Feed was withheld the morning before slaughter. Animals were killed according to the Dutch regulations for a commercial slaughterhouse. Testes were removed and testis weight was recorded and averaged for each animal. At slaughter, warm carcass weight was determined. Meat percentage was measured by the Hennessy Grading Probe (Walstra, 1987). This method routinely is used in Dutch slaughterhouses to classify pigs. Backfat samples were taken from the shoulder region of each carcass and stored at -20°C until analysis.
Hormone Analyses
The GnRH antibody titers were determined by studying the binding of pig sera serial dilutions to [125I]GnRH, as described by Meloen et al. (1994). Titers were expressed as percentage binding of [125I]GnRH at a given serum dilution. Luteinizing hormone concentrations were determined with a RIA as described by Van de Wiel et al. (1984). Porcine LH for use both as a reference preparation and for iodination was purchased from UCB (code H028/H pLH, Brussels). Interassay variation was 9.1%, intraassay variation was 10.6%, and the detection limit was 0.14 ng/mL. Serum testosterone levels were determined using a Coat-a-Count kit (DPC laboratories, Los Angeles, CA) with a detection limit of 0.14 pmol/mL. The coefficients of variation for intra- and interassay variability were 4 to 6% and 7 to 11%, respectively. The amount of androstenone in backfat was determined using an ELISA (R-Biopharm, Darmstad, Germany, as distributed by Ridascreen, Almere, The Netherlands). The detection limit of this ELISA was 0.1 µg/g, and intra- and interassay coefficients of variation were 0.4 to 4.5% and 2.0 to 3.9%, respectively.
Statistical Analyses
The experiment was performed in two replications, with eight and seven litters per replication. There were two strata of variation, namely litters within replications and pigs within litters. Growth performance, carcass traits, testis weight, LH, and testosterone data were analysed by restricted maximum likelihood methods, according to the following statistical model:
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where µ is the overall mean, rep is replication, litter is the random effect of litter within replications, group is the effect of treatment with a subdivision of immunocastrates into E-IM and L-IM (boar, E-IM, L-IM, surgical castrate), and error is the random error contribution with mean 0 and variance 
2.
Because we were particularly interested in antibody titers, LH levels, testosterone levels, and testis size at two specific timepoints after immunization (namely, at booster immunization and at slaughter), no special methods were applied with respect to repeated measurements. In those cases where an overall Wald’s test yielded a significant difference between groups, homogenous subsets of groups were formed by means of least significant differences and indicated in the usual way in the tables concerned. Testis weight, LH, and testosterone levels were log-transformed to reduce heterogeneity of variance. For antibody titers, only two groups were involved in the statistic analysis, so that a t-test could be applied. All significance levels were set at 5%. The REML analyses were performed with Genstat (Genstat 5 Committee, 1993, release 3, Reference manual. Clarendon Press, Oxford, UK) and the nonparametric analyses with StatXact (StatXact 4 for Windows, 1998, Cytel Software Corporation, Cambridge, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Eight weeks after the first immunization, significant antibody titers, ranging from 12 to 45% binding of [125I]GnRH in 1/2000 serum dilution, were present in 14 out of 15 immunocastrated pigs. At slaughter, high antibody titers were present in all immunocastrated pigs. There was no significant difference in antibody titers among E-IM and L-IM pigs, although antibody titers were numerically higher for E-IM pigs at both timepoints (Table 1
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), whereas LH levels in the L-IM pigs were higher (P < 0.001) than in E-IM pigs. Testosterone levels at 8 wk were lower (P < 0.05) in both L-IM and E-IM pigs than in the intact boars, but were substantially higher (P < 0.001) in L-IM (Table 1) than in E-IM pigs. There was no difference in testosterone levels at 8 wk between E-IM and surgical castrates. At slaughter, LH and testosterone levels were lower in both E-IM and L-IM pigs (P < 0.05) than in intact boars. Luteinizing hormone and testosterone levels at slaughter of the nonresponding immunized boar were 0.94 ng/mL and 6.74 pmol/mL respectively, which is in the range of intact boar values. The difference in biological response (i.e., testosterone and LH at 8 wk) did not depend on the BW of the immunized pigs at time of first immunization. Body weight was 23.1 kg and 23.0 kg for L-IM and E-IM pigs, respectively. Testis Function
The differences in LH and testosterone levels between E-IM and L-IM pigs at the time of booster immunization corresponded with the differences in testes size at that time. Testes length was smaller (P < 0.001) in E-IM pigs than in L-IM pigs (4.1 and 5.7 cm, respectively), whereas similar testis length was found in L-IM pigs and intact boars (Figure 1). At slaughter, testes of both E-IM and L-IM pigs were smaller (P < 0.001) than testes of the intact boars, whereas testes were smaller (P < 0.001) in E-IM than L-IM pigs. This corresponded with a lower (P < 0.001) testis weight for the E-IM pigs than for L-IM pigs (17 g and 41 g, respectively), while testis weight of the intact boars was 163 g. When analyzed for all immunocastrated pigs, the correlation between testosterone concentrations at 8 wk and the size of the testis (r = 0.85 ; P < 0.01) was much higher than the correlation between GnRH antibody titer and testis size (r = -0.42; P < 0.05).
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Androstenone concentrations in backfat of all immunocastrated and surgically castrated pigs were below the detection limit of the assay (<0.1 µg androstenone/g backfat). In the intact boars, androstenone levels in backfat ranged from undetectable to 1.25 µg/g and mean value was 0.48 µg/g. Androstenone concentration in the backfat of the nonresponding immunized pig was 0.19 µg/g.
Growth Performance
Average daily gain for the period between the first (d 0) and the second immunization, 8 wk later, was not influenced by treatment (P = 0.17), although a difference between boars and E-IM was observed. For the second period (8 wk to slaughter), and the whole experimental period (d 0 to slaughter), differences among treatments were more pronounced (Table 2). Boars grew faster than immunocastrates or surgical castrates (P < 0.05) between 8 wk and slaughter. For the whole experimental period, ADG of the L-IM pigs was less than that of the intact boars (P = 0.02), and higher (P < 0.05) than the ADG of the E-IM pigs or surgical castrates.
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). After the second immunization, the FE of both E-IM and L-IM pigs was similar to that of the surgical castrates and lower than that of the boars (P < 0.01). Overall, FE was higher in L-IM pigs than in surgical castrates or E-IM pigs (P < 0.05), but lower than in boars (P = 0.001). Carcass Characteristics
Backfat thickness at 90 kg BW, was less for L-IM pigs than for E-IM pigs or surgical castrates (P < 0.01; Table 3), and did not differ from intact boars. At slaughter, backfat thickness of the boars and L-IM pigs was thinner than for the E-IM pigs (P < 0.03), but was no longer different from the surgical castrates. Meat percentage was inversely proportional to backfat thickness at slaughter, which also was reflected in the higher meat percentage in boars and L-IM pigs (P < 0.01) as compared to E-IM pigs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Androstenone levels were undetectable in the backfat of both E-IM and L-IM pigs, demonstrating that a complete clearance of androstenone from the fat tissue was achieved, a process that is known to require a period of at least 3 wk (Claus et al., 1994). In the present study, androstenone levels exceeded the threshold value of 0.5 µg androstenone/g backfat in 50% of the intact boars, whereas the average androstenone value was 0.48 µg/g. In other studies, similar figures were found (Bonneau et al., 1994; Hennessy et al., 1997; Walstra et al., 1999).
Mean testis weight of the intact boars was relatively low (163 g) compared to results of other studies (Falvo et al., 1986; Meloen et al., 1994). This may be due to the relatively early age of the pigs at slaughter, which was 158 d. At this age, testis weights are rapidly increasing (FlorCruz and Lapwood, 1978; Van Straten and Wensing, 1978; Lunstra et al., 1986). Most likely, a majority of the pigs in our study did not reach this stage. Despite the young age of the pigs, a distinct difference in testis size at slaughter could be observed between immunocastrated pigs and boars (Figure 1). Testes of the E-IM and L-IM pigs were 50% and 25% smaller, respectively, than testes of the boars. The difference in testis size between boars and immunocastrated pigs also became visible by the outside appearance of the scrotum. Immunocastrates exhibited a flat scrotal sac, while the scrotum of the boars had a bulbous appearance. This enables us to distinguish immunocastrated pigs from intact boars by the size of the testis and the appearance of the scrotum.
In the present study, the results for ADG under restricted feeding conditions corroborate with values in the literature (Walstra and Vermeer, 1993). However, differences in FE between intact boars and surgical castrates were markedly smaller than expected. Nevertheless, FE in the L-IM pigs was higher than in the E-IM pigs and the surgical castrates (P < 0.05). An improved, more boarlike FE of immunocastrated pigs vs surgical castrates also has been reported by Bonneau et al. (1994) and Zeng et al. (2002). In both studies, however, pigs were given ad libitum access to feed. It is well known that under ad libitum feeding conditions, surgical castrates eat more than boars, subsequently resulting in a less efficient FE of the surgical castrates compared to boars. In this way the difference in FE between intact boars and surgical castrates is enlarged, which makes it more likely for FE of the immunocastrates to be between the FE of boars and surgical castrates. In our previous study (Zeng et al., 2002), this indeed was observed, despite the fact that most of the immunocastrated pigs exhibit hormone profiles similar to the E-IM pigs in the present study. In the study of Bonneau et al. (1994), the high FE of the immunocastrates may not only be due to the ad libitum feeding regimen, but also to the fact that the second immunization was applied only 2 wk before slaughter, and that the immune response of some immunized pigs was rather low. The higher FE in the L-IM pigs than in the E-IM and the surgical castrates also was reflected in a higher ADG for the L-IM pigs (P < 0.05). A comparable effect was observed under ad libitum feeding conditions (Zeng et al., 2002), where both ADG and FE tended to be higher in immunocastrates than in surgical castrates.
There were no significant differences between intact boars and surgical castrates in meat percentage and backfat thickness at slaughter. This can most likely be explained by the fact that the feed intake of the pigs was restricted, and therefore the surgical castrates deposited less fat, resulting in smaller differences for meat percentage and backfat thickness than expected under ad libitum feeding conditions (Walstra et al., 1977; Zeng et al., 2002). This also could be the reason that the L-IM pigs did not differ from the surgical castrates at slaughter. However, backfat thickness at 90 kg BW was less in the L-IM pigs than in surgical castrates, and both meat percentage and backfat thickness at slaughter were better in the L-IM pigs than in the E-IM pigs. In conclusion, growth performance was better in the L-IM pigs than in the E-IM pigs and the surgical castrates, and carcass quality was better in L-IM pigs than in E-IM pigs. Thus, "late" immunocastration could be an alternative for surgical castration, in that it not only prevents the occurrence of boar taint, but also has the advantage of improved growth performance.
A fully effective "late" immunocastration, which allows discrimination between immunocastrates and boars at slaughter, can be achieved by application of a vaccine that comprises an immunogenic GnRH antigen in combination with an adjuvant that generates a rapid and high secondary immune response. In this case, the first immunization, which is intended to prime the immune system, preferably elicits a low or moderate antibody response without GnRH-neutralizing activity. The vaccine formulation used in our previous studies (Oonk et al., 1998; Turkstra et al., 2002; Zeng et al., 2002), that is, the highly effective D-Lys6-GnRH tandem dimer ovalbumin conjugate in Specol adjuvant, does not meet these criteria. Application of the Specol water-in-oil emulsion induces a biological effect in a majority of the treated boars after a single immunization. Nevertheless, even with mineral oil adjuvants, a second immunization is needed to elicit a GnRH-neutralizing antibody response in all treated animals throughout the experiment. In practice, "late" immunocastration requires an effective second immunization, administered at least 5 wk before slaughter in order to obtain a distinct difference in testis size necessary to distinguish immunocastrated boars from untreated boars. The use of vaccines that are able to establish "late" immunocastration, due to a strong secondary response, most likely will result in improved performance of the immunized pigs.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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Received for publication January 31, 2002. Accepted for publication June 26, 2002.
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