J. Anim. Sci. 2005. 83:2901-2907
© 2005 American Society of Animal Science
Reproductive characteristics of grass-fed, luteinizing hormone-releasing hormone-immunocastrated Bos indicus bulls1
J. A. Hernandez*,
E. L. Zanella
,
R. Bogden
,
D. M. de Avila*,
C. T. Gaskins* and
J. J. Reeves*,2
* Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman 99164;
and
Universidade de Passo Fundo, Passo Fundo-RS, Brazil 99052; and
and
Amplicon Express, Pullman, WA 99163
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Abstract
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Two field trials were conducted in Brazil to evaluate LHRH immunocastration of Bos indicus bulls (d 0 = 2 yr of age). In Study I, 72 bulls were assigned randomly to one of three treatment groups: LHRH0-immunized, castrated, and intact. Immunized animals (n = 25) received a primary and two booster injections of ovalbumin-LHRH-7 and thioredoxin-LHRH-7 fusion proteins on d 0, 141, and 287. Twenty-three bulls were surgically castrated on d 141, and 24 served as intact controls. All animals were slaughtered on d 385, at approximately 3 yr of age. In Study II, 216 bulls were assigned randomly to the same three treatments as in Study I; however, because of a drought in the area, bulls were kept on pasture an additional year, and a fourth treatment was added, in which one-half the LHRH-immunized bulls received an additional booster on d 639 (fourth immunization). All animals in Study II were slaughtered on d 741 (4 yr of age). Luteinizing hormone-releasing hormone antibodies increased following each immunization for immunized bulls, but they were not detectable in castrate or intact animals in either study. Consequently, scrotal circumference was suppressed in immunized bulls compared with intact controls in both studies. By d 287, serum concentrations of testosterone in LHRH-immunized bulls were decreased compared with intact controls (P < 0.01). In both studies, testes and epididymal weights at slaughter were greater (P < 0.01) for intact (500 ± 17 and 60 ± 2 g, respectively) than for immunized bulls (173 ± 22 and 26 ± 2 g, respectively) and fourth immunization bulls (78 ± 23 and 20 ± 2 g, respectively; Study II). At the end of each study, BW was greater (P < 0.01) for intact bulls than for castrated and LHRH-immunized animals. In these two studies, the efficacy of the LHRH fusion proteins to induce an effect similar to that of surgical castration was considered 92 and 93%, respectively. These data support the concept that immunocastration of bulls at 2 yr of age was successful and that it has practical application as a tool for producing grass-fattened bulls in Brazil.
Key Words: Bos indicus • Bull • Immunocastration • Luteinizing Hormone-Releasing Hormone
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Introduction
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Active immunization against LHRH has been long recognized as a key hormonal target for preventing reproduction in livestock. Immunoneutralization of LHRH in bulls decreases LH and FSH, which are necessary for androgen production and spermatogenesis (Jeffcoate et al., 1982
; Jago et al., 1997). The subsequent decrease in testosterone leads to gonadal atrophy and impairment of reproductive function (Arimura et al., 1973; Adams et al., 1996; Miller et al., 2000). Testosterone is responsible for male characteristics, such as aggression and superior growth (Field, 1971; Seideman et al., 1982; Cosgrove et al., 1996), and ablation of testosterone by immunocastration results in an effect similar to surgical castration (Robertson et al., 1979).
Surgical castration remains an important tool in beef cattle management to decrease aggressive behavior and improve management ease; however, disadvantages associated with castration include the potential for morbidity and mortality (Schanbacher, 1982; Bonneau and Enright, 1995) and decreased growth rate (Huxsoll et al., 1998). In a country like Brazil, where hormonal implants are not allowed and the presence of screw-worms exacerbates the probability for death loss from surgical castration, bulls are left intact and grass-fattened for 3 yr. Because hormone implants are not approved in Brazil, the androgenic effect of the testes for improved growth is important. Two years of age would be the desired age at which to castrate bulls to suppress aggressive behavior because they are grazed in large groups. Given that surgical castration at this age is extremely traumatic and alternative castration methods (burdizzo or banding) are not common practices, immunocastration could be used to accomplish the goals of surgical castration without the associated death loss. We conducted two studies that were designed to examine the effectiveness of immunocastration with LHRH fusion proteins on 2-yr-old pasture-fed bulls raised in Mato Grosso, Brazil.
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Materials and Methods
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Preparation of Antigen
Fusion proteins ovalbumin-LHRH-7 and thioredoxin-LHRH-7 were prepared as previously described (Zhang et al., 1999; Quesnell et al., 2000). Ovalbumin-LHRH-7 contains seven LHRH AA sequences genetically engineered into four different positions in the ovalbumin protein, whereas thioredoxin-LHRH-7 contains seven LHRH peptide sequences genetically engineered into three distinct positions of the thioredoxin protein. The E. coli strain BL21(DE3) was used to express the proteins. Each gene construct expressed a 6-histidine AA sequence (Histag; Novagen, San Diego, CA) at the carboxyl terminus to facilitate purification of the LHRH fusion proteins via nickel affinity chromatography. Purified proteins were combined on an equimolar basis to yield a total of 1.5 mg of protein per injection. The LHRH fusion proteins were emulsified in a water-oil adjuvant containing Mycobacterium butyricum for the primary immunization and the same adjuvant without Mycobacterium butyricum for booster injections. Shipment of the anti-LHRH vaccine was approved by the Brazilian Ministry of Agriculture under document numbers 184, 01.044, 01.021/2002, and 01.021/2003.
Animals and Treatments
Nelore-cross bulls were maintained on two separate ranches in Mato Grosso, Brazil. Bulls were approximately 2 yr of age at the initiation of the study (d 0) and grazed Brachiaria brizantha pastures.
In Study I, 72 bulls (average BW = 329 ± 4.3 kg) from the Fazenda Esperança ranch were assigned randomly to one of three treatments. An intact control group (n = 24) remained untreated for the duration of the study, whereas a second group of bulls (n = 23) was surgically castrated 141 d after initiation of the study. The remaining bulls (n = 25) were immunized against the LHRH fusion proteins. Immunizations were administered as a primary injection and two s.c. booster injections on d 0, 141, and 287, respectively, in the neck. Surgical castration was performed at the time of the first booster injection (d 141). Body weight and scrotal circumference were recorded on d 0, 141, 287, and 385. Mean epididymal and testes weights were collected at the time of slaughter (d 387).
In Study II, 216 bulls (average BW = 258 ± 2.9 kg) from the Fazenda Colorado ranch were assigned randomly to one of three treatments: intact controls (n = 72), castrate (n = 72), and LHRH-immunized (n = 72). As a result of an exceptionally dry season, however, bulls in this study were kept on pasture for an additional year. Therefore, a fourth treatment group was implemented, in which an additional booster was given to 30 randomly selected bulls from the LHRH-immunized treatment (d 639; fourth immunization). The remaining LHRH-immunized bulls that did not receive the fourth immunization experienced a 454-d interval from their final booster injection until all animals were slaughtered (d 741). Body weight and scrotal circumference were recorded on d 0, 141, 287, 639, and 741. Paired epididymal and testes weights were collected at the time of slaughter (d 741). Both studies were designed to allow for slaughter after a withdrawal period of at least 90 d from the time of the last booster injection.
Blood samples for Study I were collected (coccygeal venipuncture) on d 141, 287, and 387. For Study II, blood samples were collected on d 141, 287, 639, and at slaughter (d 741). Blood samples were stored at 4°C for 24 h, and serum was decanted into plastic vials and stored at –20°C until analyses. Serum samples were treated with 0.2% (wt/vol) citric acid to lower pH and inactivate any potential Foot and Mouth virus and were then shipped to Washington State University under APHIS P No. 50322.
Hormone Analyses
Serum testosterone and LHRH antibody-binding activity were evaluated to provide an indication of vaccine efficacy. Serum testosterone was quantified by solid-phase RIA with a commercial kit from Diagnostic Systems Laboratory (Webster, TX). The average intraassay CV was 14.4%, with an interassay CV of 8.9%; the sensitivity of the assay was 0.05 ng/mL. Percentage of 125I-LHRH bound in a 1:1,000 dilution of each serum was determined using procedures described by Johnson et al. (1988). All serum samples and tubes brought into the United States were destroyed by incineration after assay analysis in fulfillment of APHIS regulations.
Statistical Analyses
Percentages of LHRH antibody binding, testosterone, and BW were analyzed using the Mixed procedure of SAS (SAS Inst., Inc., Cary, NC). The experimental design was completely randomized with repeated measures, and the model included treatment, which was tested using animal within treatment as the error term; day and treatment x day interaction, which were tested using the residual mean square. When a significant treatment x day interaction was detected (P < 0.05), treatments were compared within day. Two orthogonal contrasts were used to compare the treatments for testosterone and BW. The first compared castrated with immunized bulls, and the second compared intact with the average of castrated and immunized bulls. Small variances in castrated and immunized animals relative to intact animals necessitated use of a log10 transformation of the testosterone values. Reported means and SE from the analysis of the transformed data were converted back to the original scale by taking the anti-log. For Study I, two orthogonal contrasts were used to compare the treatments for percentage of LHRH antibody binding. The first compared intact with castrated bulls, and the second compared immunized with the average of castrated and intact bulls. For Study II, three orthogonal contrasts were used to compare the treatments for LHRH antibody binding. The first compared immunized with late-immunized bulls, the second compared castrated with the average of the immunized and late-immunized groups, and the third compared intact with the average of the castrated, immunized and late-immunized animals. All comparisons were chosen before the study and compared the groups with the same expected biological response for specific traits. Scrotal circumference and epididymal and testes weights were subjected to an analysis of variance for a one-way completely random design, and least squares means are reported.
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Results
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Study I
Body Weight.
A treatment x day interaction was detected for BW (P = 0.01). On the first day of the study (d 0), the 2-yr-old bulls weighed 329 ± 4.3 kg. Body weight among the three treatment groups did not differ (P = 0.71; Table 1) 141 d after initiation of the study. By d 287, however, BW was greater (P = 0.02) for intact controls (472 ± 7.4 kg) than for castrated and LHRH-immunized animals (451 and 452 ± 7.7 kg, respectively). Two days before slaughter (d 385), intact bulls continued to be heavier (P < 0.01), at 523 ± 7.4 kg vs. 485 ± 7.6 kg and 486 ± 7.3 kg for castrated and immunized animals, respectively.
Testes Measurements.
A treatment x day interaction was detected for scrotal circumference (P = 0.01). Scrotal circumference was similar between intact and LHRH-immunized bulls on d 0 (P = 0.74); however, by d 141, circumference tended to differ (P = 0.06) for intact and immunized groups (Figure 1). Intact bulls continued to demonstrate greater scrotal circumference than immunized bulls on d 287 and at the time of slaughter (d 385; P < 0.01). Paired epididymal and testes weights were measured at the time of slaughter as an indicator of vaccine effectiveness (d 387; Table 2). Epididymal weights for intact bulls averaged 47 ± 1.0 g compared with 25 ± 1.1 g for the immunized bulls. Similarly, testes weights for intact bulls were greater (P < 0.01) than those for LHRH-immunized bulls. Two bulls in the LHRH-immunized group were identified as nonresponders to the vaccine as a result of a scrotal circumference measurement >29 cm at the time of slaughter.
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Figure 1. Mean scrotal circumference (right axis) of intact (- -) or LHRH-immunized (-•-) bulls over 385 d in Study I. Immunizations against LHRH were administered at times indicated by arrows, and breaks in the x-axis represent unequal time intervals. Bars indicate LHRH binding activity (left axis) of immunized animals presented by percentage of 125I-LHRH bound at a 1:1,000 serum dilution.
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Table 2. Paired testes and epididymal fresh tissue weights for intact and LHRH-immunized bulls at slaughter on d 387 for Study I and d 741 for Study IIa
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LHRH Antibody Binding and Serum Testosterone.
Antibody binding activity for LHRH was influenced by the treatment x day interaction (P = 0.01). Antibody binding of LHRH was greater (P < 0.01) in immunized bulls by d 141 compared with castrated or intact animals (Figure 1
). Percentage of LHRH binding increased over time (P < 0.01) in the LHRH bulls, but was undetectable at all time points for intact or castrated bulls. Immunized animals that received a final booster injection 100 d before slaughter (d 287) had mean LHRH antibody binding of 31 ± 1.8% at slaughter.
Testosterone concentrations varied because of the treatment x day interaction (P < 0.01; Table 3). At the time of the first booster immunization (d 141), serum concentrations of testosterone were greater for intact bulls than for immunized animals (P < 0.01). Castration of bulls on d 141, after measurements were collected, resulted in decreased serum concentrations of testosterone. Following the third immunization (d 287), mean testosterone concentrations for LHRH-immunized animals remained decreased compared with intact bulls, but their testosterone was greater than that of castrated animals (P = 0.01). However, by d 387 (slaughter), bulls immunized against LHRH had testosterone concentrations similar to those of the castrate group (P = 0.10)
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Table 3. Concentrations of testosterone (ng/mL) in serum from LHRH-immunized, castrated, or intact bulls during 385- or 741-d studiesa
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Study II
Body Weight.
A treatment x day interaction was detected for BW (P < 0.01). On d 0, the average BW was 258 ± 2.9 kg. As in Study I, BW did not differ (P = 0.33; Table 1) up to 141 d after initiation of the study; however, BW on d 287 was greater (P < 0.01) for intact controls than for castrated and LHRH-immunized animals. By d 639, when the fourth treatment group was implemented, the intact bulls weighed 513 ± 5.2 kg compared with 456 ± 4.9 kg for castrated animals and 455 and 458 (±6.8 kg) for immunized and fourth immunization bulls, respectively. Two days before slaughter (d 741), intact bulls continued to be heavier than castrated animals and both LHRH-immunized groups (P < 0.01).
Testes Measurements.
Scrotal circumference was similar between intact and LHRH-immunized bulls on d 0 (P = 0.90); however, by d 141, intact bulls demonstrated a greater rate of increase in scrotal circumference than immunized bulls (P < 0.01; Figure 2). Intact bulls demonstrated a steady increase in scrotal circumference throughout the study. Suppression in scrotal circumference growth was observed for LHRH-immunized bulls on d 141, 287, and 385. A further decrease in scrotal circumference was observed following the final booster injection (d 639) for bulls receiving four immunizations. At the end of the study (d 741), scrotal circumference was 35.4 ± 0.47 cm for intact bulls and 23.0 ± 0.61 and 19.9 ± 0.64 cm for the immunized and fourth immunization groups, respectively. As in Study I, paired epididymal and testes weights were collected at the time of slaughter (d 741). Epididymal and testes weights of intact bulls were heavier (P < 0.01) than those from both LHRH-immunized groups (Table 2); however, bulls immunized with three injections had greater epididymal and testes weights than did those that received the fourth immunization.
LHRH Antibody Binding and Serum Testosterone.
A treatment x day interaction (P < 0.01) was detected for LHRH antibody binding. Luteinizing hormone-releasing hormone antibody binding activity was detected by d 141 in immunized bulls, and the percentage binding increased following each LHRH immunization throughout the duration of the study (Figure 2). Castrated and intact animals did not display antibody binding at any point during the 741-d study. At the time the fourth treatment group was implemented (d 639), LHRH antibody binding was 29.6 ± 2.0% for immunized bulls and 28.5 ± 2.1% for fourth immunization bulls (P > 0.72). Antibody binding activity increased on d 741 for bulls receiving the fourth immunization compared with immunized bulls that did not receive the additional injection (39.4 ± 2.2 and 24.0 ± 2.2%, respectively).
A treatment x day interaction (P < 0.01) was detected for serum testosterone concentrations; therefore, treatments were examined within day (Table 3). Testosterone concentrations did not differ among the treatment groups on d 141 (P > 0.10); however, on d 287, intact bulls had greater concentrations of testosterone than the other treatment groups (P < 0.01). Intact bulls continued to demonstrate greater concentrations of circulating testosterone for the duration of the study. Testosterone concentrations for immunized bulls were greater than concentrations for castrated animals at all time points from d 287 to 741 (P < 0.05). The optimal time for use of this immunocastration protocol would be during the last 9 mo before slaughter with the last booster 90 d before slaughter.
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Discussion
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The results of the present studies indicate that the LHRH-7 fusion proteins are effective in inducing an immunogenic response to LHRH in Bos indicus cattle at 2 yr of age. In both studies, immunization against LHRH resulted in sufficient antibodies against LHRH to alter testes development and function. Although differences between assays limit comparisons of antibody binding between studies from different labs, LHRH-immunized bulls had antibody activities (31.7 and 24% binding for Studies I and II, respectively) at the end of each study that exceeded the classification of a good response described by Adams et al. (1996), where good was designated as >10% 125I-LHRH binding at a 1:1,000 serum dilution. In both studies, bulls immunized against LHRH at 2 yr of age demonstrated decreased testis size and serum testosterone concentrations by 3 yr of age compared with 3-yr-old control bulls.
Antibody binding activity of LHRH remained greater for immunized bulls than for either intact or castrated animals at the end of Studies I and II. In early studies by Robertson et al. (1982), bull calves immunized against conjugated LHRH showed suppression of gonadal function and sexual behavior that was reversed after only 6 mo. However, the results of Study II are more comparable with those observed in the long-term suppression of testicular function in 20 to 25% of ram lambs immunized at an early age (Brown et al., 1994), and 16 to 35% of sexually mature Bos indicus bulls (D’Occhio et al., 2001). In Study II, 72% of bulls receiving a final booster injection 15 mo before slaughter continued to exhibit levels of LHRH antibody binding activity that resulted in sustained small (<29 cm) scrotal circumferences and decreased serum testosterone. Additionally, 9% of these animals demonstrated no detectable LHRH antibody binding, but they had scrotal circumferences <29 cm. These bulls were not expected to return to a preimmunization state, and they might have developed nonreversible lesions in the median eminence. This type of lesion has been demonstrated in boars that had the highest response to an immunization against LHRH (Molenaar et al., 1993).
Animal growth and development are influenced in large part by the gonadal steroids (Schanbacher, 1984). Removal of testicular function by castration or immunization against LHRH was associated with a marked decrease in growth rate in both of the present studies. By d 287 of both studies, intact bulls had begun to gain BW more rapidly than both surgically and immunocastrated animals. Intact bulls continued to demonstrate superior growth compared with the castrate groups for the duration of each study. The decreased growth rate observed in the castrated and LHRH-immunized bulls compared with intact bulls is similar to results described in steers and in immunized bulls by Huxsoll et al. (1998), and it suggests that the anabolic effects of testosterone are eliminated in LHRH-immunocastrated bulls. Bulls immunized against LHRH at 2 yr of age would be expected to demonstrate larger growth and more weight gain than animals castrated at an earlier age, as observed by Aïssat et al. (2002). Carcass performance data previously published on a representative group of bulls from Study I (de A. Ribeiro et al., 2004) demonstrated that immunized bulls were comparable with surgically castrated steers, with increased marbling, backfat, and decreased LM area compared with the intact bulls.
Decreased scrotal circumference and testes weights in LHRH-immunized bulls are consistent with those previously reported in bulls (Huxsoll et al., 1998; Cook et al., 2000; Aïssat et al., 2002). These data support the earlier findings that LHRH immunocastration suppresses reproductive function, as demonstrated by regression of the gonads (Arimura et al., 1973). Adams et al. (1996) suggested that in the US beef industry, 7 mo of age may be the optimal age for immunization against LHRH for maximal antibody production and the most pronounced suppression of testicular weight in Bos taurus bulls slaughtered 11 mo after a single primary immunization. However, in both studies herein, 92 and 93% of bulls immunized at 2 yr of age and receiving a booster injection 3 mo before slaughter maintained growth rate and serum testosterone concentrations similar to those of castrated animals. When a 15-mo interval existed between the last booster injection and the time of slaughter, testes and epididymal weights, along with testosterone concentrations, had begun to rebound to control bull levels. Therefore, for maximal suppression of reproduction, the final booster injection should be delivered up to 90 d before slaughter.
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Footnotes
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1 Research was supported by a Washington Technology Center Grant, National Research Initiative Competitive Grant no. 2000-35206-93551 and 2003-35203-13514 from the USDA Cooperative State Research, Education, and Extension Service, 00-35206-9355 to J. J. Reeves and Amplicon Express, Pullman, WA. Sincere thanks are expressed to J. and K. Carter, owners of Fazenda Esperança, for their generous hospitality and for making this study possible by allowing us to use their facilities and bulls. Appreciation is extended to H. Hugo of Fazenda Colorado for use of his bulls and facilities. Thanks also to C. Small for help in preparing the proposal and assistance in working cattle and to J. Kleberg for assistance in working the cattle. 
2 Correspondence: P. O. Box 641063 (phone: 509-335-8339; fax: 509-335-4246; e-mail: reevesjj{at}wsu.edu).
Received for publication September 13, 2004.
Accepted for publication July 5, 2005.
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Abstract
Introduction
-) over 741 d in Study II. Immunizations against LHRH were administered at times indicated by arrows, and breaks in the x-axis represent unequal time intervals. Bars indicate LHRH binding activity (left axis) of LHRH-immunized bulls receiving three immunizations (open bars) or four immunizations (hatched bar) presented by percentage of 125I-LHRH bound at a 1:1,000 serum dilution.