The effects of active immunization against GnRH on testicular development, feedlot performance, and carcass characteristics of beef bulls 1

Transcription

1 The effects of active immunization against GnRH on testicular development, feedlot performance, and carcass characteristics of beef bulls 1 R. B. Cook*, J. D. Popp, J. P. Kastelic 2, *, S. Robbins, and R. Harland *Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB T1J 4B1; Manitoba Department of Agriculture, 1129 Queen Ave., Brandon, MB, R7A 1L9; and Biostar Inc. 343, 111 Research Dr., Saskatoon, SK, S7N 3R2 ABSTRACT: The objective was to determine the effects of a recombinant fusion protein anti-gnrh vaccine on testicular development, feedlot performance, and carcass quality of beef bulls. Crossbred beef bulls (n = 58, average weight 306 kg, 9 mo of age), were randomly allocated to two groups and received either an anti-gnrh vaccine (GnRH) or placebo (Control) by intramuscular injection on d 0, 56, and 112. There were group effects (P < 0.01; as a percentage of Control) on testicular weight (53%), daily sperm production (40%), and epididymal sperm reserves (16%). There were group time interactions (P < ) for scrotal circumference and serum testosterone concentrations; at slaughter, bulls in the GnRH group had a smaller (P < 0.05) scrotal circumference (28.3 vs 33.9 cm) and lower (P < 0.05) serum testosterone concentrations (2.2 vs 8.6 ng/ml) than those in the Control group. Average daily gain, feed intake, and feed efficiency were not different between treatments during the backgrounding phase (d 0 to 84). During the finishing phase (d 98 to 182), ADG was greater (P < 0.05) for bulls in the Control group (1.69 vs 1.42 kg/d), as was carcass weight (6.9%; P < 0.01). However, GnRH bulls had numerically better feed efficiency (6.12 vs 7.08 kg DMI/kg gain; P < 0.23) and shear force values for ribeye that were 16% lower (P < 0.14) than Control bulls, warranting further investigation. Vaccinating bulls against GnRH suppressed testicular function, with growth and carcass characteristics similar to that expected with steers. Key Words: Bulls, Carcasses, Castration, GnRH, Immunological Techniques, Testosterone 2000 American Society of Animal Science. All rights reserved. J. Anim. Sci : Introduction Immunoneutralization of the hypothalamic decapeptide GnRH retards testicular growth and inhibits spermatogenesis (Schanbacher, 1984; Deaver et al., 1988) and is an alternative to surgical castration (Robertson et al., 1979). Testicular growth and maturation is regulated by GnRH (Amman, 1983); appropriate regulation 1 Lethbridge Research Centre Contribution No Appreciation is expressed to Biostar Inc. for their support, including providing funds, cattle, vaccine, and conducting assays. Financial support from the Agriculture and Agri-Food Canada s Matching Investment Initiative is gratefully acknowledged. The authors wish to thank D. Giacchetta, C. Cockwill, and R. Wilde for technical assistance and I. Walker and staff for feeding and care of livestock. Our gratitude is expressed to Cargill Foods Ltd. for their cooperation in slaughtering the bulls and the Canadian Beef Grading Agency for carcass data collection. Portions of these data were presented at the American Society Animal Science national meeting in Denver, Colorado, July, Correspondence: P.O. Box 3000 (phone: 403/ ; fax: 403/ ). Received December 21, Accepted June 1, of this peptide can modulate serum testosterone concentrations, thereby maintaining good growth performance while reducing carcass masculinity (Adams et al., 1993). Immunized bulls have greater carcass weights than steers and lower masculinity scores than bulls not immunized (Adams and Adams, 1992; Adams et al. 1993). Responses seemed optimal when immunization occurred before puberty. Bull calves have marked increases in GnRH secretion after 4 mo of age (coincident with increases in LH secretion), at which time onset of prepubertal transition and testicular development begins (Rodriguez and Wise, 1989, 1991). However, beneficial effects on carcass development and testicular growth have resulted from even a single immunization at 4 to 12 mo of age (Adams et al., 1996). To date, development of an efficacious, yet practical, GnRH antigen has faced problems of ineffective immunization and(or) extensive purification processes that increase cost (Manns et al., 1997). One alternative strategy is to use a carrier protein to generate a strong antibody response (Manns et al., 1997). The objective of the current study was to determine the effect of a new recombinant fusion protein anti- GnRH vaccine on reproductive development, feedlot performance, and carcass quality of beef bulls. 2778

2 GnRH immunization of bulls 2779 Table 1. Composition of backgrounding and finishing phase diets Item Background Finish Ingredient, DM basis, % Barley grain Barley silage Supplement Nutrient composition, DM basis, % CP ADF NDF Starch DE, Mcal/kg Materials and Methods Fifty-eight composite (crossbred) beef bulls (mean BW, ± 6.2 kg; 9 mo of age) were ranked by BW and scrotal circumference and randomly assigned to receive an intramuscular injection of either recombinant fusion protein anti-gnrh vaccine (GnRH; n = 28; Manns et al., 1997) or placebo (Control; n = 30). Injections were administered in the neck, immediately anterior to the shoulder and dorsal to the cervical vertebrae. The anti-gnrh vaccine was produced by Biostar Inc. (Saskatoon, SK, Canada). A carrier protein leukotoxin, produced by Pasteurella haemolytica, was used to generate a strong antibody response. A chimeric protein was formed by ligating an oligonucleotide synthesized to code for the peptide GnRH (eight copies, fused to both the carboxy and amino ends of the leukotoxin carrier protein) and then produced through a highly regulated bacterial expression system (Manns et al., 1997). The product, a recombinant fusion protein anti-gnrh vaccine, was administered with a commercially acceptable adjuvant. The time of the initial injection was designated d 0, with subsequent injections given on d 56 and d 112. Bulls were fed a backgrounding diet (d 0 to d 84); then, after a 14-d feed adaptation period, bulls were fed a finishing diet (d 98 to 182) as outlined in Table 1. Bulls were used and cared for in accordance with guidelines of the Canadian Council on Animal Care (Olfert et al., 1993). Every 14 d, access to water was withdrawn overnight (approximately 14 h), and bulls were weighed in the morning. Feed efficiency was determined on a per-pen basis; there were four pens per treatment, two containing 11 bulls and two containing four bulls. Unconsumed feed was weighed weekly (to determine total feed consumed for the period) and then discarded. Feed samples were collected every 7 d to determine DM and chemical composition. Feed samples were dried and ground to pass through a 1-mm screen (Wiley Mill) and analyzed for moisture (AOAC, 1990, method 7.007), Kjeldahl nitrogen (method 7.021), starch (Herrera-Saldana et al., 1990), and acid and neutral detergent fiber (Van Soest et al., 1991). On d 0, 70, 98, 126, 154, 168, and 182, blood samples were collected by jugular venipuncture. Analysis of serum testosterone concentrations (GammaCoat, Testosterone 125 I RIA Kits, INCSTAR Corp., Stillwater, MN) and anti-gnrh titers (Meloen et al., 1994) were determined by radioimmunoassay in duplicate samples. For the testosterone assay, the limit of detection was ng/ml and the intra- and interassay coefficients of variation were 6.2 and 7.7%, respectively. On d 0, 28, 56, 84, 98, 126, 154, and 182, scrotal circumference was measured with a consistent-tension scrotal tape (Coulter Scrotal Tape, Trueman Mfg., Edmonton, AB, Canada). Bulls were sent to slaughter on d 183; carcass data recorded included warm carcass weight, average fat cover (three measurements of fat thickness overlying the longissimus muscle) and longissimus muscle area (these three variables were measured manually and subsequently used to determine the percentage of lean meat yield), and quality grade (Agriculture Canada, 1992). Shear force was measured on longissimus muscle samples (seven bulls per group, randomly chosen prior to initial immunization) with a Warner-Bratzler shear cell (Mir et al., 1997). Testes were retrieved from these same seven bulls in each group. Testes and epididymides were weighed and tissue samples were collected for determination of daily sperm production, epididymal sperm reserves (Amann et al., 1973), and histological examination (Cook et al., 1994). The average most-advanced stage of spermatogenesis was determined by observing 20 independent microscopic fields per bull at 200 using bright-field microscopy. Stages were ranked 1 to 5 according to the following categories: Stage 1, A and B1 spermatogonia; Stage 2, B2 intermediate and zygotene spermatogonia; Stage 3, pachytene primary spermatocytes; Stage 4, spermatids from steps one through nine of spermiogenesis; and Stage 5, steps 10 to 14 of spermiogenesis (Berndston and Desjardins, 1974). Histological evaluations were done in the blind (without knowledge of bull identity or treatment group). A GLM repeated measures analysis (SAS Institute Inc., Cary, NC) was used to determine main effects of group and time, and the group time interactions for serum testosterone concentrations, anti-gnrh titers, scrotal circumference, BW, ADG, feed intake, and feed efficiency. If the effect of group or the group time interaction was significant, Student s t-test was used to evaluate the difference between group means for each measurement day. For both groups, linear correlations were calculated between all end points and stepwise regression analyses were performed to determine bestfitting linear regression models for several end points as dependent variables. For each regression, independent variables were considered for inclusion if they were correlated (P < 0.15) with the dependent variable. To avoid intercorrelation, if two candidate independent variables were correlated (P < 0.15), then only the independent variable that was more highly correlated with

3 2780 Cook et al. Table 2. Serum testosterone concentration and anti-gnrh titer of 58 beef bulls that received either an anti-gnrh vaccine (GnRH) or placebo (Control) on d 0, 56, and 112. A group time interaction (P < ) was observed for both end points Day of observation Group Testosterone concentration, ng/ml GnRH b 5.4 b 0.5 b 2.4 b 2.2 b Control c 9.1 c 7.0 c 10.5 c 8.6 c SEM a Anti-GnRH titer, 1/1,000 dilution GnRH d 63.1 d 50.6 d 77.1 d 72.4 d 69.8 d Control e 0.1 e 0.3 e 1.4 e 1.4 e 1.3 e SEM b,c Within a column, means with unlike superscripts differ (P < 0.05). d,e Within a column, means with unlike superscripts differ (P < ). the dependent variable was included in the regression analysis. Results and Discussion A group time interaction (P < ) was detected for serum testosterone concentrations (Table 2). Following the second immunization on d 56, mean serum testosterone concentrations in the two groups were similar on d 70 but were lower (P < 0.05) in the GnRH group than in the Control group on d 98. Following the third immunization (d 112), mean testosterone concentrations were further reduced; concentrations were below the limit of detection in 75% of the GnRH group for the last three samples. However, seven GnRH bulls had testosterone concentrations similar to those of the Control bulls toward the end of the trial, indicating some variability in immune response. In that regard, SEM for serum testosterone concentrations appeared to increase throughout the study and final testosterone concentrations had a negative linear regression (r 2 = 0.22; P < 0.02) with final anti-gnrh titers. Therefore, bulls with the highest titers had the lowest testosterone concentrations. Scrotal circumference (Table 3) increased steadily throughout the study in the Control bulls. However, in bulls in the GnRH group, scrotal circumference reached a plateau at d 98 and was smaller (P < 0.05) than in Control bulls from d 84 to the end of the study (group time interaction, P < ). Scrotal development was initially similar in the two groups; however, in immunized bulls, further development was suppressed, concurrent with reductions in serum testosterone concentrations. In that regard, final testosterone concentrations accounted for 35% of the variation in final scrotal circumference in GnRH bulls (P < 0.001). The testes and epididymides weighed less (P < 0.001) in the GnRH group than in the Control group (Table 4), consistent with the smaller scrotal circumference; similar results were found by Adams et al. (1993) and Jeffcoate et al. (1982). Total daily sperm production (Table 4) was severely reduced in the immunized bulls (P < 0.01). However, the reduction in daily sperm production per gram of testicular parenchyma did not reach significance (P < 0.09), due, in part, to the large variation in daily sperm production in the immunized bulls (range, 0.6 to 18.4 million cells/g). In a previous study (Adams et al., 1993), daily sperm production was lower in bulls immunized against GnRH compared with control bulls (7.6 vs 24.6 million cells/g, respectively). Final anti-gnrh titers of the immunized bulls accounted for approximately 75% of the variation in both Table 3. Scrotal circumference (cm) of 58 beef bulls that received either an anti-gnrh vaccine (GnRH) or placebo (Control) on d 0, 56 and 112. A group time interaction (P < ) was detected Day of observation Group GnRH b 29.2 b 29.3 b 28.7 b 28.3 b Control c 30.9 c 32.2 c 32.9 c 33.9 c SEM a b,c Within a column, means with unlike superscripts differ (P < 0.05).

4 GnRH immunization of bulls 2781 daily sperm production per gram (P < 0.03) and total daily sperm production (P < 0.02). A positive linear regression (r 2 = 0.75) was observed (P < 0.02) between final testosterone concentrations and epididymal weight in bulls in the GnRH group. Furthermore, the regression model for total epididymal sperm reserves in the immunized bulls had a negative linear regression with final testosterone concentration (P < 0.001) and tended (P < 0.06) to have a positive linear regression with final anti-gnrh titers. Therefore, immunized bulls with reduced testosterone concentrations had a smaller epididymis containing fewer spermatozoa. No significant regression models were observed for Control bulls. The average most advanced stage of spermatogenesis observed was lower (P < 0.04) for the immunized bulls (Table 4); approximately 35% of the these bulls had no detectable elongated spermatids (Stage 5). Although immunization did not cause sterility, it profoundly suppressed spermatogenesis. The anti-gnrh vaccine suppressed serum testosterone concentrations in the majority (75%) of vaccinated bulls, with subsequent effects on testicular size (scrotal circumference) and sperm production ability. Similar results are reported in the literature (Adams and Adams, 1992; Adams et al., 1996). It is noteworthy that Robertson et al. (1979, 1982, 1984) and Gonzalez et al. (1990) reported that a maximum of 50% of bulls immunized against GnRH responded with increased antibody titers, reduced testosterone concentrations, and decreased testicular size. Warm carcass weight (Table 4) was greater (P < 0.01) in the Control group than in the GnRH group (352.4 vs kg respectively); lean meat yield, however, was nearly identical (62.3 vs 62.9%), in agreement with Gonzalez et al. (1990) and Finnerty et al. (1996). Anti- GnRH titers tended (P < 0.06) to have a negative linear regression (r 2 = 0.14) with longissimus muscle area, suggesting that the greater the response to the vaccine, the more steerlike the carcass. The slaughter results may be partially confounded by slaughtering all bulls following a common number of days on feed rather than at a common degree of finish. However, it is believed that similar carcass results would have been reported had the slightly lighter GnRH bulls been fed for a longer interval (e.g., 14 d). The majority of bulls from both groups were finished at the time of slaughter, as indicated by fat around the tail head and the tightness of the paunch (general visual appearance); this was reflected in the lack of treatment differences in average fat cover of the carcass (Table 4). Shear value (Table 4) was not different between treatment groups (5.27 vs 6.27, P < 0.14). However, testosterone concentrations of immunized bulls had a positive linear regression with shear value (r 2 = 0.77; P < 0.01), suggesting that the greater the response to GnRH immunization, the more tender the meat. There was one grade AAA carcass (GnRH group) and one dark cutter (Control group), with the remaining bulls grading approximately 42% grade AA and 55% grade A across groups. Adams et al. (1996) reported no difference in carcass dressing percentage between treatment groups, with a concurrent reduction in carcass masculinity (as assessed by the USDA grading system). That there was only a single dark cutter may have been due to the bulls being accustomed to frequent handling and a minimal interval (approximately 3 h) from the feedlot to slaughter. Minimal growth performance differences occurred between the two groups during the backgrounding phase (Table 5). During the finishing phase, Control bulls gained more weight (P < 0.05) than those in the GnRH group (Table 5). The reduction in ADG in the vaccinated bulls could be partially attributed to a reduced feed intake (P < 0.06), although the differences in ADG were not large (P > 0.05). Other studies (Adams et al., 1993, 1996) have reported that control bulls had Table 4. Effect of an anti-gnrh vaccine on reproductive and carcass characteristics at slaughter Item GnRH Control SEM a P value b Testis wt, g Epididymal wt, g Spermatogenesis c DSP d /g, 10 6 cells DSP total, 10 6 cells ESR e total, 10 9 cells Carcass wt, kg Longissimus muscle area, cm Fat, mm Lean yield, % Shear value f b P value = observed significance level. c Spermatogenesis = average most advanced stage of spermatogenesis (ranked 1 to 5). d DSP = daily sperm production. e ESR = epididymal sperm reserves. f Shear value = tenderness as determined using Warner-Bratzler shear cell.

5 2782 Cook et al. Table 5. Effect of an anti-gnrh vaccine on growth performance during backgrounding (d 0 to 84) and finishing (d 98 to 182) phases in 58 beef bulls that received either an anti-gnrh vaccine (GnRH) or placebo (Control) Item GnRH Control SEM a P-value Body weight for backgrounding, kg Initial Final Body weight for finishing, kg Initial Final Overall ADG, kg/d Backgrounding phase Finishing b Overall feed intake, DM basis, kg/d Backgrounding Finishing Overall feed efficiency, DMI kg/kg gain Backgrounding Finishing b Within the row, means differ (P < 0.05). slightly higher feedlot gains than immunized bulls but have not reported improved feed efficiency. Implications Immunizing young bulls against GnRH suppressed serum testosterone concentrations, testicular development, and spermatogenesis. Growth and carcass characteristics were similar to those expected with steers, with indications of improved tenderness. More research is needed to reduce the variability in response to the vaccine and to determine the optimal timing of vaccination. Literature Cited Adams, T. E., and B. M. Adams Feedlot performance of steers and bulls actively immunized against gonadotropin-releasing hormone. J. Anim. Sci. 70: Adams, T. E., C. A. Daley, B. M. Adams, and H. Sakurai Testis function and feedlot performance of bulls actively immunized against gonadotropin-releasing hormone: Effect of implants containing progesterone and estradiol benzoate. J. Anim. Sci. 71: Adams, T. E., C. A. Daley, B. M. Adams, and H. Sakurai Testes function and feedlot performance of bulls actively immunized against gonadotropin-releasing hormone: Effect of age at immunization. J. Anim. Sci. 74: Agriculture Canada Livestock carcass grading regulations. Canada Gazette. Part II. 126:3821. Amman, R. P Endocrine changes associated with onset of spermatogenesis in Holstein bulls. J. Dairy Sci. 66: Amman, R. P., J. F. Kavanaugh, L. C. Griel, and J. K. Voglmayr Sperm production of Holstein bulls determined from testicular spermatid reserves, after cannulation of rete testis or vas deferens, and by daily sperm production. J. Dairy Sci. 57: AOAC Association of Official Analytical Chemists Official Methods of Analysis. 15th ed. Washington, DC. Berndston, W. E., and C. Desjardins The cycle of the seminiferous epithelium and spermatogenesis in the bovine testis. Am. J. Anat. 140: Cook, R. B., G. H. Coulter, and J. P. Kastelic The testicular vascular cone, scrotal thermoregulation, and their relationship to sperm production and seminal quality in beef bulls. Theriogenology 41: Deaver, D. R., J. D. Glass, D. M. Grieger, and J. J. Reeves Effects of estradiol on secretion of LH, hypothalamic function and testicular development in bull calves. Domest. Anim. Endocrinol. 5: Finnerty, M., W. J. Enright, D. J. Prendiville, L. J. Spicer, and J. F. Roche The effect of different levels of gonadotropinreleasing hormone antibody titres on plasma hormone concentrations, sexual and aggressive behaviour, testis size and performance of bulls. Anim. Sci. (Penicuik) 63: Gonzalez, A., S. Goubau, A. F. Allen, R. J. Mapletoft, R. Cohen, and B. D. Murphy The potential for castration of domestic animals by active immunization against GnRH. Livestock Reproduction in Latin America. pp FAO-IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna, Austria. Herrera-Saldana, R. E., J. T. Huber, and M. H. Poore Dry matter, crude protein, and starch degradability of five cereal grains. J. Dairy. Sci. 73: Jeffcoate, I. A., J. M. S. Lucas, and D. B. Crighton Effects of active immunization of ram lambs and bull calves against synthetic luteinizing hormone releasing hormone. Theriogenology 18: Manns, J. G., C. Barker, and S. K. Attah-Poku The design, production, purification, and testing of a chimeric antigen protein to be used as an immunosterilant in domestic animals. Can. J. Chem. 75: Meloen, R. H., J. A. Turkstra, H. Lankhof, W. C. Puijk, W. M. M. Schaaper, G. Dijkstra, C. J. G. Wensing, and R. B. Oonk Efficient immunocastration of male piglets by immunoneutralization of GnRH using a new GnRH-like peptide. Vaccine 12: Mir, P. S., D. R. C. Bailey, Z. Mir, S. D. M. Jones, T. Entz, S. D. Husar, N. H. Shannon, and W. M. Robertson Effect of feeding barley based diets on animal performance, carcass characteristics and meat quality of crossbred beef cattle with and without Wagyu genetics. Can. J. Anim. Sci. 77: Olfert E. D., B. M. Cross, and A. A. McWilliam Canadian Council of Animal Care Guide to the Care and Use of Experimental Animals. 2nd ed. vol. 1. Brada Printing Services, Ottawa, ON. Robertson, I. S., H. M. Fraser, G. M. Innes, and A. S. Jones Effect of immunological castration on sexual and production characteristics in male cattle. Vet. Rec. 111:

6 GnRH immunization of bulls 2783 Robertson, I. S., J. C. Wilson, and H. M. Fraser Immunological castration in male cattle. Vet. Rec. 105: Robertson, I. S., J. C. Wilson, H. M. Fraser, G. M. Innes, and A. S. Jones Immunological castration of young bulls for beef production. Curr. Top. Vet. Med. Anim. Sci. 26: Rodriguez, R. E., and M. E. Wise Ontogeny of pulsatile secretion of gonadotropin-releasing hormone in the bull calf during infantile and pubertal development. Endocrinology 124: Rodriguez, R. E., and M. E. Wise Advancement of postnatal pulsatile luteinizing hormone secretion in the bull calf by pulsatile administration of gonadotropin-releasing hormone during infantile development. Biol. Reprod. 44:432. Schanbacher, B. D Pituitary-testicular responses of estradiol- 17β-implanted bull calves to continuous versus pulsatile infusion of luteinizing hormone releasing hormone. J. Anim. Sci. 58: Van Soest, P. J., J. B. Robertson, and B. A. Lewis Methods for determining dietary fibre, neutral detergent fibre, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74: