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J. Anim. Sci. 2002. 80:2209-2213
© 2002 American Society of Animal Science

Endocrine, growth, and carcass characteristics of bulls immunized against luteinizing hormone-releasing hormone fusion proteins1

D. Aïssat*, J. M. Sosa*,{ddagger}, D. M. de Avila*, K. P. Bertrand{dagger} and J. J. Reeves*,{ddagger},2

* Department of Animal Sciences, and {dagger} School of Molecular Biosciences, and and {ddagger} Center for Reproductive Biology, Washington State University, Pullman 99164-6353

2 Correspondence:
ASLB 220 (phone: (509) 335-8339; fax: (509) 335-7354; E-mail:
jjreeves{at}wsu.edu).


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
This study evaluated the effectiveness of a LHRH fusion protein vaccine on endocrine changes, feedlot performance, and carcass quality of bulls compared with steers and hormone-implanted steers. Crossbred bulls (n = 30; mean weight, 179 ± 4 kg; mean age, 130 ± 2 d) were randomly assigned to three treatment groups: 1) castrated (castrated; n = 10); 2) castrated-implanted with trenbolone acetate (implanted; n = 10); and 3) immunized against a cocktail of recombinant fusion proteins, ovalbumin-LHRH-7 and thioredoxin-LHRH-7 (immunized bulls; n = 10). Blood was collected every 2 wk to evaluate antibody and hormone concentrations. Serum LHRH antibodies (P < 0.001) were detected in animals of the immunized group, which had reduced serum LH concentrations (P < 0.001) compared with the castrated groups and serum FSH concentrations, which did not decrease but were significantly different when compared with castrated and implanted animals. Serum testosterone concentrations in the immunized bulls were not different from the two castrated groups (P > 0.05) by d 60 after primary immunization. Initial mean scrotal circumference of the immunized bulls was 18.0 ± 0.6 cm on d 0 and increased to 22.6 ± 1.3 cm by d 310. No differences (P > 0.05) in ADG were observed among treatment groups. Immunized animals had an intermediate BW gain (P > 0.05) when compared with the castrates, whereas the castrated groups differed (P < 0.05) from each other. Carcass characteristics were similar (P < 0.05) among the three groups. Vaccinating bulls against a LHRH fusion protein cocktail suppressed LH and testosterone, which led to reduced testicular development and no bullock carcasses. Growth and carcass characteristics of the immunized animals were similar to the steers.

Key Words: Bulls • Carcass • Growth • Immunization • LHRH


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Scientists have realized the potential use of immunocastration as a viable alternative to physical castration for decades. This can be accomplished through vaccination to block reproductive hormones involved in testicular function such as LH, FSH, and LHRH. Arimura et al. (1973) first described the action of LHRH vaccination on rabbit testes during development of antibodies for use in an LHRH RIA. Although various laboratories have tested LHRH vaccines for immunocastration (Schanbacher, 1984; Adams and Adams, 1986; Reeves et al., 1989) in various species, no commercial product has become available in the United States. A major problem with first generation (conjugated) LHRH vaccines is meeting the Food and Drug Administration regulatory requirements for a homogenous preparation of antigen. An additional problem became evident with Vaxstrate (Hoskinson et al., 1990), a conjugated ovalbumin LHRH vaccine, which was marketed in Australia from 1990 to 1995 and has since been taken off the market. Cattle producers were unsatisfied with results since a substantial number of heifers continued to cycle. Ideally, the vaccine should be at least 95 to 100% effective. In an attempt to overcome these problems, different laboratories have developed recombinant LHRH vaccines (Van der Zee et al., 1995; Zhang et al., 1999; Hsu et al., 2000) that were shown to be effective and were chemically defined as required by the FDA.

Quesnell et al. (2000) reported that a combination of two recombinant LHRH fusion proteins, ovalbumin-LHRH-7 and thioredoxin-LHRH-7, appeared superior to either fusion protein alone in significantly reducing gonadal function when tested in male mice. The present study evaluated the effectiveness of a combination of these two LHRH fusion proteins on immunocompetence in bulls fed and slaughtered as feedlot steers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
Animals and Treatments
Thirty Angus and Angus cross bull calves, averaging 130 ± 2 d of age and 179 ± 4 kg BW, were randomly assigned to three treatment groups: 1) castrated (n = 10); 2) castrated-implanted (n = 10); and 3) immunized (n = 10). However, one castrated animal was removed from the study due to illness and was not included in any of the results. Surgical castration was performed on d 0 of the experiment. Implanted steers received a s.c. Finaplix-H (200 mg trenbolone acetate) on d 28 and 163.

A primary immunization and two booster injections were given on d 0, 28, and 163, respectively. Immunizations were given on alternating sides of the neck subcutaneously. Each immunization was distributed over three different injection sites. All procedures performed in this study were approved by the Washington State University Institutional Animal Care and Use Committee (LARC #1780).

Preparation of Antigen and Immunization
Immunized bulls received a mixture of two LHRH fusion proteins purified from inclusion bodies of overexpressed E. coli strains. Ovalbumin-LHRH-7 contains a total of seven LHRH sequences inserted at four different positions in the sequence of chicken ovalbumin (Zhang et al., 1999). Thioredoxin-LHRH-7 similarly contains a total of seven LHRH sequences inserted at three positions in the sequence of E. coli thioredoxin (Quesnell et al., 2000). Both LHRH fusion proteins have a 6-histidine sequence (His-Tag; pET Manual, Novagen, 1994) at the carboxyl terminus to facilitate purification by affinity chromatography as described previously (Quesnell et al., 2000). Equimolar amounts of each LHRH fusion protein (20 nM) totaling 1.5 mg of protein were suspended in 6 M urea and emulsified in 1 mL of modified complete Freund’s adjuvant with Mycobacterium butyricum (CalBiochem, San Diego, CA) for the first immunization and incomplete Freund’s adjuvant for the subsequent two boosters.

Data and Blood Sample Collection
All animals were bled via coccygeal venipuncture once every 2 wk. Blood samples were refrigerated overnight at 4°C, and then centrifuged at 2,500 x g for 15 min. Serum was stored at -20°C until subsequent analyses. All animals were weighed every 28 d. Scrotal circumference measurements were taken every 2 wk for each immunized animal.

A radioactive binding assay was used to evaluate the percentage of 125I-LHRH that would bind to the anti-LHRH antibody present in the serum at a 1:1,000 dilution (Johnson et al., 1988). Serum LH, FSH (Adams et al., 1975; Acosta et al., 1983), and testosterone (Diagnostic Systems Laboratories Inc., Webster, TX) concentrations were determined by RIA. The interassay coefficients of variation were 7.6%, 20%, and 21% respectively. The standard preparations were USDA-bLH-B6 and USDA-bFSH-B1 for the LH and FSH assays, respectively.

Diets and Feed Analysis
Feed samples were collected weekly for feed analysis. All animals consumed 6 kg/d alfalfa and triticale hay ad libitum during the backgrounding period, which consisted of the first 212 d of the study. During the finishing period, which spanned the last 98 d of the study, animals were fed 1.8% hay (50% alfalfa and 50% triticale hay), 7.5% alfalfa haylage, 87.2% rolled barley, and 3.5% supplement (50% cull peas, 33% barley, 6.4% Bovatec (Roche Vitamins, Inc., Parsippany, NJ), 6.2% trace mineral salt with selenium, 3.1% selenium premix, 1% animal fat, and 0.3% vitamin A premix (30,000 IU/g)) on a DM basis.

Carcass Data
All animals were slaughtered on d 312 at a commercial abattoir at a mean age of 444 ± 2 d and a mean weight of 554 ± 9 kg. They were slaughtered after a 90-day withdrawal period from the last booster injection as specified by the Investigational New Animal Drug Approval #10575. Carcass data including carcass weight; marbling score; ribeye area; yield grade; kidney, pelvic, and heart fat (KPH); back fat; and carcass maturity were collected 24 h postmortem according to USDA grading standards (USDA, 1997).

Statistical Analysis
Data analysis was performed using the Mixed procedure of SAS (SAS Inst. Inc., Cary, NC) for repeated measures to determine main effects of treatment, time, and treatment x time for each of the response variables (serum LH, serum FSH, serum antiLHRH antibody percentage bound, serum testosterone, BW, and average daily gain). The GLM procedure of SAS was used for all carcass data analysis. Scrotal circumference was evaluated using time as the main effect.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
AntiLHRH antibodies were first detected by d 42 in immunized bulls (P < 0.001, Figure 1AGo). The LHRH binding activity increased steadily from d 42 and reached a plateau after d 181 (after the second booster) with 71 ± 1% bound at a 1:1,000 dilution. Neither steer group had detectable amounts of LHRH antibodies at any time during the trial.



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  		Figure 1. A) Mean ± standard error of antibody binding to LHRH expressed as a percentage bound 125I-LHRH 1:1,000 dilution in LHRH immunized bulls, steers, and trenbolone acetate-implanted steers fed to slaughter. Solid arrows represent days of immunizations and open arrows represent days to trenbolone acetate implantation (SEM ± 5.47, 0, and 0, respectively). B) Mean serum concentrations of LH in the LHRH immunized bulls, steers, and trenbolone acetate-implanted steers fed to slaughter (SEM ± 0.10, 0.18, and 0.05, respectively). C) Mean serum concentrations of FSH in the LHRH immunized bulls, steers, and trenbolone acetate-implanted steers fed to slaughter. Solid arrows represent days of immunizations. Open arrows represent days of implant (SEM ± 3.27, 3.23, and 0.27, respectively). D) Mean serum concentrations of testosterone in the LHRH immunized bulls, steers, and trenbolone acetate-implanted steers fed to slaughter (SEM ± 0.08, 0.08, and 0.10, respectively).

 
Serum concentrations of LH of the immunized bulls were reduced (P < 0.001) by the immunization and were low until the end of the trial when compared with steers in the castrated-nonimplanted group, which had the highest serum LH concentration (Figure 1BGo). Serum concentrations of FSH were not reduced by the immunization but were different (P < 0.05) from steers in both castrated groups. Serum testosterone concentrations in the immunized bulls were not different from testosterone concentrations in the steers (P > 0.05, Figure 1DGo).

Scrotal circumference for immunized bulls ranged from 19.0 to 22.4 cm for nine animals. The remaining animal had a scrotal circumference of 33.5 cm on the last day of the study, resulting in a group mean of 22.6 ± 1.3 cm on d 310 compared with their initial mean measurement of 18 ± 0.6 cm on d 0. These bulls averaged 22.7 ± 1.0 cm at 12 mo of age which corresponds to d 180 of the study.

Animals were evaluated for two distinct feeding periods, backgrounding and finishing. During both periods, the ADG of the immunized bulls was intermediate to the mean values for castrated and implanted animals (Table 1Go). Weight gained during the study was highest (P < 0.05) for both the castrated and implanted compared with the castrated animals (Table 1Go).


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  		Table 1. Weight gain and carcass data for steers, implanted steers, and LHRH immunized bulls
 
None of the carcasses in the immunized or the implanted groups graded as bullocks, nor did any of the immunized bulls’ carcasses exhibit the presence of injection site granulomas. The LHRH immunization did not compromise mean marbling score, yield grade, or ribeye area when compared with carcasses from steers in both castrated groups (Table 1Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
Results from this study demonstrate that immunization against LHRH reduces serum LH and testosterone concentrations, as well as testicular size. This is consistent with the results of previous studies (Adams et al., 1996; Huxsoll et al., 1998; Cook et al., 2000). The LHRH immunized animals in this study had carcass characteristics intermediate to the two steer groups. Average daily gain of the LHRH immunized bulls was not significantly different from the steers. However, the nonimplanted steers had lower total weight gain over the treatment period than the implanted steers or the immunized bulls. The maintenance of the BW gain in the immunized bulls similar to the implanted steers is probably associated with the residual serum concentrations of testosterone noted in the immunized cattle.

Sosa et al. (2000) found that immunization of heifers using only the ovalbumin-LHRH-7 emulsified in Zmax adjuvant (Zonagen, Inc., Woodlands, TX) induced a biologically active immune response that was reduced by a nonresponsive second booster. This may be explained by carrier-mediated immune suppression (Sad et al., 1991). Carrier-mediated immune suppression is a result of an overwhelming secondary response to the carrier protein, which inhibits the response to the hapten portion of the vaccine. The use of two carrier proteins simultaneously in this study may help to overcome high antibody production against the carrier protein. With two carriers, the immune system is not exposed to a large amount of one protein at one time. Thus, the system is able to respond to LHRH, producing antibody concentrations which peak and remain elevated after the second booster. Biological response was maintained as shown by suppressed testicular growth throughout the study. The other possible reason for the increased success of this vaccine may be due to the use of modified Freund’s adjuvant vs Zmax adjuvant. In the past, Freund’s adjuvant repeatedly proved to be consistent and effective in our studies.

The results of the present study further support the success of two LHRH fusion proteins in combination developing high levels of antibody-binding activity and subsequent biological response. Quesnell et al. (2000) using the same fusion protein combination also observed reduced testicular/anterior prostate and vesicular gland weights as well as high levels of antibody-binding activity in mice. Immunized bulls in the present study showed little increase in scrotal size on average from 18.3 ± 0.6 cm on d 0 to 22.6 ± 1.3 cm on d 310. This increase occurred mainly in the beginning of the study prior to detection of increased concentrations of LHRH antibodies. Scrotal circumference did not increase from the second booster through the end of the study. For a bull to pass a breeding soundness exam at 12 mo of age, scrotal circumference must be at least 30 cm (Ball et al., 1983). One immunized bull had a delayed response to the immunization, which may have led to the larger scrotal circumference.

Numerically, immunized bulls tended to be closer to implanted steers for carcass weight, ADG during both backgrounding and finishing, and total weight gained when compared with unimplanted steers. On the contrary, marbling scores for immunized bulls were closer to the unimplanted steers when compared with implanted animals. These observations support results previously published by Hoxsell et al. (1998), in which immunized bulls were also similar to steers. The present study shows that immunization with an LHRH fusion protein vaccine may be a viable alternative to surgical castration in livestock management.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
Immunization of bulls with a cocktail of LHRH fusion proteins was effective in altering the endocrine profile of the bulls resulting in circulating testosterone concentrations similar to steers. Carcass weights and carcass quality traits of LHRH immunocastrated and surgically castrated animals were not different. These data support the hypotheses that immunization against an LHRH fusion protein may have potential utility as a noninvasive alternative to surgical castration and at the same time may allow for the elimination of hormone implants without reduced body weight gain.


   Footnotes
 
1 Acknowledgments: A. F. Parlow, Director of National Hormone and Pituitary Program for LH and FSH used as standards in the RIA. Leo Reichert, Jr., Albany Medical School for LH and FSH used for iodination. Hoechst Roussel Vet for donating the trenbolone acetate implants. Supported by USDA grant # 03251 and Washington Technology Center grant # 01-84 to JJR and KPB. Back

Received for publication January 15, 2002. Accepted for publication April 22, 2002.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


Acosta, B., G. K. Tarnavsky, T. E. Platt, D. L. Hamernik, J. L. Brown, H. M. Schoenemann, and J. J. Reeves. 1983. Nursing enhances the negative effect of estrogen on LH release in the cow. J. Anim. Sci. 57:1530–1536.[Abstract/Free Full Text]

Adams, T. E., and B. M. Adams. 1986. Gonadotrope function in ovariectomized ewes actively immunized against gonadotropin-releasing hormone (GnRH). Biol. Reprod. 35:360–367.[Abstract]

Adams, T. E., C. A. Dailey, B. M. Adams, and H. Sakurai. 1996. Testes function and feedlot performance of bulls actively immunized against gonadotropin-releasing hormone: Effect of age at immunization. J. Anim. Sci. 74:950–954.[Abstract]

Adams, T. E., J. E. Kinder, P. K. Chakraborty, V. L. Estergreen, and J. J. Reeves. 1975. Ewe luteal function influenced by pulsatile administration of synthetic LHRH/FSHRH. Endocrinology 97:1460–1467.[Abstract]

Arimura, A., H. Sato, T. Humasaka, R. B. Wonobec, L. Debeljak, J. Dunn, and A. V. Schally. 1973. Production of antiserum to LH-releasing hormone (LHRH) associated with gonadal atrophy in rabbits: Development of radioimmunoassays for LHRH. Endocrinology 93:1092–1103.[Medline]

Ball, L., R. S. Ott, R. G. Mortimmer, and J. C. Simmons.1983. Manual for breeding soundness examination of bulls. J. Soc. Theriogenology XII, Society of Theriogenology, Hastings, NE. pp 1–65.

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Huxsoll, C. C., E. O. Price, and T. E. Adams. 1998. Testis function, carcass traits, and aggressive behavior of beef bulls actively immunized against gonadotropin-releasing hormone. J. Anim. Sci. 76:1760–1766.[Abstract/Free Full Text]

Hsu, C., C. Ting, C. Ting, T. Chen, C. Lin, J. Whang-pen, and J. Hwang. 2000. Vaccination against gonadotropin-releasing hormone (GnRH) using toxin receptor-binding domain-conjugated GnRH repeats. Cancer Res. 60:3701–3705.[Abstract/Free Full Text]

Johnson, H. E., D. M. de Avila, C. F. Chang, and J. J. Reeves. 1988. Active immunization of heifers against luteinizing hormone-releasing hormone, human chorionic gonadotropin, and bovine luteinizing hormone. J. Anim. Sci. 66:719–726.[Abstract/Free Full Text]

pET System Manual. 1994. Novagen, Inc., Madison, WI.

Quesnell, M. M., Y. Zhang, D. M. de Avila, K. P. Bertrand, and J. J. Reeves. 2000. Immunization of male mice with LHRH fusion protein reduces testicular and accessory sex gland function. Biol. Reprod. 63:347–353.[Abstract/Free Full Text]

Reeves, J. J., C. F. Chang, D. M. de Avila, D. M. Grieger, H. E. Johnson, and A. J. Roberts. 1989. Vaccines against endogenous hormones: A possible future tool in animal production. J. Dairy Sci. 72:3363–3371.[Abstract/Free Full Text]

Sad, S., H. M. Gupta, G. P. Talwar, and R. Raghupathy. 1991. Carrier-induced suppression of the antibody response to a ‘self’ hapten. Immunology 74:223–227.[Medline]

Schanbacher, B. D. 1984. Active immunization against LHRH in the male. In: B. D. Crighton, (ed.) Immunological Aspects of Reproduction in Mammals. pp 345–362. Butterworth, London.

Sosa, J. M., Y. Zhang, D. M. de Avila, K. P. Bertrand, and J. J. Reeves. 2000. Technical Note: Recombinant LHRH fusion protein suppresses estrus in heifers. J. Anim Sci. 78:1310–1312.[Abstract/Free Full Text]

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