Use of recombinant gonadotropin-releasing hormone antigens for immunosterilization of beef heifers.

The objectives of this study were to evaluate the effects of immunization against recombinant GnRH fusion proteins and growth promotants on onset of puberty, feedlot performance, and carcass characteristics of beef heifers. Heifers were immunized against an ovalbumin fusion protein containing 7 GnRH peptides (oGnRH, n = 12), a thioredoxin fusion protein containing 7 GnRH peptides (tGnRH, n = 12), a combination of oGnRH plus tGnRH (otGnRH, n = 12), or a combination of ovalbumin and thioredoxin (control, n = 11). Each heifer received a primary immunization containing 1 mg of protein in 1 mL of adjuvant injected into the mammary gland at wk 0 (mean age = 38 wk) and booster immunizations at wk 6 and 12. Six heifers within each treatment received Synovex H implants at wk -2. Weekly blood samples were collected from wk -2 to 26 for determination of serum progesterone concentrations and GnRH antibody titers. In GnRH-immunized heifers, GnRH antibody titers increased after the first booster injection, peaked after the second booster injection, and remained elevated through the end of the study (P < 0.01). Heifers immunized against oGnRH achieved greater (P < 0.05) GnRH antibody titers than tGnRH heifers but did not differ (P = 0.20) from otGnRH heifers. During the 26-wk study, ovulation was prevented (P < 0.05) in 10 out of 12, 12 out of 12, 11 out of 12, and 0 out of 11 tGnRH, oGnRH, otGnRH, and control heifers, respectively. At slaughter, uterine weights were lighter (P < 0.01) for GnRH-immunized heifers than control heifers. Synovex H-implanted heifers had greater (P < 0.05) ADG from wk -2 to 26, greater LM area, and lesser percentages of KPH, yield grade, and quality grade than nonimplanted heifers, regardless of the immunization treatment. Immunization against GnRH fusion proteins resulted in production of antibodies against GnRH that prevented ovulation in 92% of the heifers without affecting feedlot or carcass performance. Implanting heifers with Synovex H improved ADG, LM area, and yield grade. Improvements in delivery of the oGnRH vaccine may provide a feasible alternative to surgical spaying of heifers.

Reproductive characteristics of grass-fed, luteinizing hormone-releasing hormone-immunocastrated Bos indicus bulls.

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.

Changes in spermatogenesis and endocrine function in the ram testis due to irradiation and active immunization against luteinizing hormone-releasing hormone.

Spermatogonial stem cell transplantation is a technique that has potential in livestock to enhance genetic gain and generate transgenic offspring through the male germ line. A means for depletion of endogenous germ cells in a recipient's seminiferous tubules is necessary for this technology to be applied. The objectives of this study were to evaluate several methods for depletion of endogenous germ cells in the testes of adult rams and to evaluate ultrasound-guided injections into the rete testes as a means for infusing a suspension into the seminiferous tubules. Sixteen adult rams were randomly divided into 4 treatment groups (n = 4 per group). Treatments consisted of active immunization against LHRH (IMM), localized testicular irradiation (IR), LHRH immunization + irradiation (IMM+IR), and untreated control. Serial bleedings were conducted pretreatment and monthly after treatment for 4 mo, at which time all rams were castrated. Both IMM and IMM+IR rams received exogenous gonadotropin in the form of Perganol weekly for 8 wk before castration to bypass the immunization. All rams also received an ultrasound-guided injection of PBS containing 0.4% trypan blue into the rete testis of one testicle before castration. Rams receiving IMM and IMM+IR treatments had higher (P < 0.05) average percentages of seminiferous tubule cross sections with depleted germ cells compared with controls. Serum testosterone was decreased (P < 0.05) in IMM and IMM+IR rams 1 mo after treatment and throughout the remainder of the study compared with controls and IR rams, which were not different from each other. Serum inhibin concentration was unchanged in all rams following treatment indicating that Sertoli cell function was unaltered. A greater (P < 0.05) average percentage of the total testicular area could be filled with the trypan blue solution by rete testis injection in IMM and IMM+IR rams. These data demonstrate the depletion of endogenous germ cells in adult ram testes without alteration of Sertoli cell viability and function that have potential as methods for preparing recipient animals for germ cell transplantation.

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

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.

Transplantation of bovine germinal cells into mouse testes.

To develop techniques for spermatogonial transplantation in bulls, it is essential to have an effective bioassay procedure to evaluate the transplantation efficiency of spermatogonial stem cell collection, purification, and culture techniques. The objective of the present study was to develop a mouse bioassay model to evaluate transplantation efficiency of fresh and cultured bovine germ cells. Bull calves of four ages (1, 2, 3, and 4 mo) were used as a source of donor testes cells. Two calves were used for each age point, one calf was experimentally made cryptorchidistic at 1 wk of age and the other left normal. A STO (mouse fibroblast) feeder cell line was used to culture bovine testes cells for 2 wk preceding transfer into recipient testes. Immunodeficient nude mice (nu/nu) in which endogenous spermatogenesis had been abolished by busulfan treatment served as recipient animals for transplantation. Donor bovine germ cells were microinjected into mouse seminiferous tubules. Mouse testes were analyzed 2 wk after transplant with the use of a bovine-specific antibody and whole-mount immunohistochemistry for the presence of bovine donor germ cells. Bovine testis cells were present in all recipient mouse testes analyzed. Fresh bovine testes cells were observed as colonies of round cells within mouse seminiferous tubules, indicating spermatogonial expansion and colonization; however, cultured bovine testes cells appeared as fibrous tissue and not as spermatogenic colonies. The average number of colonies resulting from donor cryptorchid testes was not different (P > 0.05) from noncryptorchid, 56+/-4 and 78+/-7, respectively. Fresh donor cells from calves older than 1 mo gave rise to a greater average number of colonies within recipient testes (P <0.05) (1 mo, 33+/-4; 2 mo, 70+/-8; 3 mo, 63+/-6; 4 mo, 87+/-9). Fresh bovine germ cells are capable of colonization in the busulfan-treated nude mouse testis, making it a suitable model for evaluation and development of spermatogonial transplant techniques in bulls.

LHRH antagonist decreases LH and progesterone secretion but does not alter length of estrous cycle in heifers.

The objective of this study was to evaluate the suppressive effect of an LHRH antagonist, Cetrorelix SB-75 (SB-75), on secretion of LH, FSH and ovarian function in beef heifers. In Exp. 1, heifers were treated with a single dose of 10 microg/kg body weight intramuscularly on d 3 of the estrous cycle. In Exp. 2, heifers received either a single injection (100 microg/kg) of SB-75 on d 3 of the estrous cycle or multiple injections of 20 microg/kg on d 3, 4, 5, 6, and 7. Serum LH, but not FSH, was suppressed from one to several days. However, neither FSH nor progesterone was significantly altered. In Exp. 3, heifers received an injection vehicle (5% mannitol) or 100 microg/kg BW of SB-75 on d 1 of the estrous cycle (30 h after first observed standing estrus). Injection of SB-75 suppressed LH pulse frequency on d 3, 5, and 7 (P < 0.001). The mean LH concentrations in the SB-75 treatment groups were lower on d 3 (P < 0.01) and 5 (P < 0.05). There were no differences (P > 0.1) between the two groups in the mean concentrations of LH on d 1, 7, or 14. Treatment did not affect the secretion pattern or concentration of FSH. Injection of SB-75 did not alter estradiol-173 concentrations (P > 0.1). Treatment reduced corpus luteum (CL) function as indicated by lower progesterone production. However, the length of the estrous cycle was not shortened. These data show that the CL can form and survive in the face of depressed LH concentrations during the early stages of the estrous cycle.

The effects of recombinant LHRH fusion proteins on testicular development and histology in ram lambs.

Two recombinant luteinizing hormone releasing hormone (LHRH) fusion proteins were evaluated for their effectiveness in suppression of testicular development and histology by injecting together. Recombinant fusion proteins, ovalbumin-LHRH-7 and thioredoxin-LHRH-7, were generated using recombinant DNA technology and expressed in E. Coli. Eleven ram lambs ranked by age and body weight were randomly assigned to receive either ovalbumin and thioredoxin recombinant protein mixture (control group, n = 5) or ovalbumin-LHRH-7 and thioredoxin-LHRH-7 recombinant fusion protein mixture, anti-LHRH vaccine, (immunization group, n = 6). Animals in each group were weaned at 17 wk of age and were injected (primary immunization) with either mixture at 18 wk of age. Both groups received a booster immunization 8 wks later (26 wk of age). Scrotal circumference, scrotal length, testicular diameter and testicular length were measured in both groups every other week. All animals were slaughtered at 36 wk of age. Immediately after slaughter, a small testicular tissue was taken and processed for histological examination. In the ram lambs in immunization group scrotal circumference and testicular diameter increased steadily until second booster and then remained as a plateau until the end of the experiment. The differences in scrotal circumferences and testicular diameter were significant between the two groups during the last three weeks of the study (p < 0.05). There were no differences in testicular and scrotal length throughout the study (p > 0.05). Seminiferous tubules lost their regular shape and were decreased in diameter in immunization group. Although a few spermatozoa were seen in some tubules, in general, there were atrophy of the seminiferous tubules and loss of spermatogenesis, nevertheless, it seemed that animals in this group were potentially fertile.

Immunization of male mice with luteinizing hormone-releasing hormone fusion proteins reduces testicular and accessory sex gland function.

Genes for ovalbumin-luteinizing hormone-releasing hormone 7 (LHRH-7) and thioredoxin-LHRH-7 fusion proteins (containing seven LHRH inserts) were constructed by cassette and mismatch mutagenesis and expressed in Escherichia coli. In experiment 1, 10 microgram of either ovalbumin-LHRH-7 or thioredoxin-LHRH-7 were suspended in Z-max adjuvant and injected three times at 4-wk intervals into postpubertal male BALB/c mice. In experiment 2, the fusion proteins were suspended in Immumax adjuvant and administered in equimolar quantities (0.4 nmol per injection) to postpubertal male BALB/c mice. In addition to injection of these two proteins alone, the proteins were also administered in different sequences or together in a mixture. Both LHRH fusion proteins induced significant antibody titers, which resulted in a significant decrease in vesicular gland and anterior prostate weight (measure of biological response) in both experiments. Vesicular gland and anterior prostate weight and LHRH antibody titers were significantly correlated in experiments 1 (r = -0.64) and 2 (r = -0.53). Percentage of animals responding to treatment varied from 40-60% in experiment 1 and from 11-89% in experiment 2, with the highest responses in treatments that used a combination of both fusion proteins. The variation in responders and nonresponders was evaluated by estimating antibody K(D) from displacement curves. Part, but not all, of the high antibody nonresponders can be explained by antibody affinity.

Technical note: recombinant LHRH fusion protein suppresses estrus in heifers.

A recombinant luteinizing hormone-releasing hormone (LHRH) fusion protein was evaluated for its effectiveness in suppression of estrus in heifers. Eight heifers were randomly assigned to two equal treatment groups. Treatments consisted of recombinant ovalbumin-LHRH-7 or recombinant ovalbumin (control). This recombinant chimeric fusion protein consisted of ovalbumin with seven LHRH peptides (ovalbumin-LHRH-7). The plasmid for this protein was expressed in E. coli and was collected and purified as an insoluble protein. One milligram of the respective proteins was suspended in 2 mL of Z-Max adjuvant and administered by intramammary injection three times at 7-wk intervals. Luteinizing hormone-releasing hormone antibody binding was elevated in heifers treated with ovalbumin-LHRH-7 compared to ovalbumin-treated heifers (P < .05). Serum progesterone concentrations (< 1 ng/mL) indicate that the estrous cycle of the four heifers treated with ovalbumin-LHRH-7 was suppressed for a time period ranging from 60 to 238 d, which was different from control heifers (P < .01). Serum progesterone for the control heifers continued to exhibit cyclic profiles over the experimental period. This preliminary study in heifers demonstrated that a chimeric LHRH fusion protein induced elevated concentrations of circulating LHRH antibodies that suppressed estrus for an average of 122 +/- 41 d.

Late-gestation treatment of pregnant cows with trenbolone acetate does not increase subsequent growth of heifer calves.

Fifty crossbred cows (38 multiparous and 12 nulliparous) were used to evaluate in utero androgenization of heifer calves with trenbolone acetate. Three 200-mg trenbolone acetate (Finaplix-H) implants were implanted in the ear of treated cows (n = 24) on d 214 +/- 11 of gestation; the remaining animals (n = 26) were used as controls. Cows' rate of gain, serum levels of trenbolone acetate, gestation length, degree of dystocia, percentage bred back, days until conception, and 24-h milk production were evaluated. Fourteen-day weigh periods until parturition indicated that trenbolone acetate-treated dams had an increased (P < .05) average daily gain (1.05 +/- .1 kg) compared with control cows (.55 +/- .1 kg). Serum concentrations of trenbolone acetate were higher (P < .01) in treated cows with a peak at 9 d after implantation and returned to basal concentrations by d 77. Treatment did not affect degree of dystocia among all cows (P > .05) but seemed to increase (P < .01) the incidence of dystocia in nulliparous cows compared with nulliparous control cows. Gestation length and subsequent fertility were not affected by treatment (P > .05). Similarly, there was no difference in 24-h milk production (P > .05) between treated and control cows. Calf birth weight, phenotypic measurements at birth, vigor, average daily gain, carcass characteristics, and heifer reproductive tract and ovarian weights did not differ with treatment (P > .05). These data showed that late-gestation treatment with 600 mg of trenbolone acetate significantly increased weight gain of dams without demonstrating any androgenizing effects on the growth or physical characteristics of heifer calves.

Trucking stress at breeding does not lower conception rate of beef heifers.

The goal of this study was to determine whether 1 h of trucking stress before or after artificial insemination (AI) altered the conception rate of beef heifers. Estrus was synchronized in heifers with prostaglandin F(2alpha). The 3 treatment groups consisted of 1) AI (control heifers, n = 93); 2) Truck + AI (trucked for 1 h immediately before AI, n = 81); and 3) AI + Truck (trucked for 1 h immediately after AI, n = 82). All heifers were artificially inseminated by a single technician with semen from a single ejaculate. Blood samples were collected for cortisol measurement 1 h before AI, immediately before and after AI, and 1 h after AI in the AI (n = 6), Truck + AI (n = 9), and AI + Truck (n = 8) groups Pregnancy in heifers was confirmed either at slaughter or by palpation per rectum. Trucking before AI elevated (P < 0.01) serum cortisol concentrations. Artificial insemination alone increased (P < 0.01) serum cortisol concentrations in AI heifers. The increase in serum cortisol concentrations caused by trucking after AI was not significant (P > 0.05). Areas under the cortisol curves in Truck + AI heifers are greater (P < 0.05) than in AI heifers. The conception rates of AI heifers (50.5%), Truck + AI heifers (51.9%) and AI + Truck heifers (58.5%) are not different (P > 0.05). This study demonstrates that 1 h of trucking stress either before or after AI did not lower the conception rate of heifers.

Antibodies against cortisol block suppressive effects of corticosteroids on lymphocytes in vitro.

Lymphocyte transformation assays were used to test the ability of antibodies against cortisol to reduce bioactivity of corticosteroids in vitro. Mononuclear cells were separated from whole bovine blood and cultured in the presence of PHA alone, PHA + steroid, PHA + steroid + anticortisol, or PHA + steroid + anti-bovine serum albumin. Tritiated thymidine uptake was determined for all groups during the last 24 hr of a 72-hr culture period by scintillation counting. Polyclonal anticortisol against cortisol-bovine serum albumin conjugated in the 21 position was more effective in blocking cortisol activity than monoclonal anticortisol built against conjugates in the 3 position. The steroids that suppressed PHA-induced lymphocyte proliferation in a concentration-dependent manner were: cortisol, corticosterone, dexamethasone, prednisolone, 11-deoxycortisol, and 11-deoxycorticosterone. Aldosterone, cortisone, cholesterol, estradiol, and progesterone did not exhibit concentration-dependent effects and, thus, were not considered suppressive. These concentration-independent steroids were also the least suppressive (with the exception of aldosterone). Anticortisol was able to reduce bioactivity of suppressive corticosteroids that had an 11-hydroxy group, suggesting the antibody was primarily made against this site. Anti-BSA was not effective in blocking corticosteroid activity, but it did enhance proliferation of lymphocytes if added in combination with weakly suppressive steroids. Anticortisol also had an enhancing effect when added with some weakly suppressive steroids. We conclude that antibodies against cortisol are capable of reducing bioactivity of steroids that strongly suppress lymphocyte proliferation. Additionally, the 11-hydroxy group may be an important antigenic determinant of steroid molecules.