Effects of immunization against luteinizing hormone-releasing hormone and treatment with trenbolone acetate on reproductive function of beef bulls and steers.

The objectives of this study were 1) to evaluate the ability of trenbolone acetate (TBA) administered in tandem with LHRH immunization to suppress reproductive function in bulls and 2) to examine the effects of LHRH and androgen (TBA) signaling on pituitary gland function. Forty-four Angus × Hereford crossbred calves (BW=225 ± 2 kg; age=187 ± 6 d) received castration, LHRH immunization, or TBA administration in a 2 × 2 × 2 factorial design. Treatment groups receiving LHRH immunization contained 6 animals, whereas other treatment groups contained 5 animals. Animals immunized against LHRH received a primary injection and 2 booster injections of ovalbumin-LHRH-7 fusion protein on d 0, 42, and 196, respectively. Animals treated with TBA were implanted on d 224. Serum LHRH antibodies increased (P<0.05) after each booster for immunized animals, but were negligible in nonimmunized animals throughout the experiment. Serum testosterone concentration (P<0.001) and scrotal circumference (P<0.05) were depressed in LHRH-immunized bulls compared with nonimmunized bulls by d 84 and 168 of the experiment, respectively. Treatment with TBA tended (P=0.08) to decrease serum testosterone concentrations of nonimmunized bulls. Weights of testes at slaughter were decreased (P<0.001) for LHRH-immunized (232 ± 41 g) compared with nonimmunized (752 ± 45 g) bulls, but did not differ (P=0.80) between TBA-implanted (500 ± 49 g) and nonimplanted bulls (484 ± 36 g). Both LHRH immunization and castration decreased pituitary gland stores of LH and FSH (P<0. 001). There was no effect (P>0.10) of TBA on pituitary gland FSH content and only a tendency (P=0.09) to increase pituitary gland LH content. Immunization against LHRH decreased expression of LH ß-subunit and common α-subunit genes (P<0.001). Castration increased expression of LH ß-subunit and common α-subunit genes (P=0.02). Treatment with TBA further suppressed (P=0.04) α-subunit mRNA expression in LHRH-immunized steers. In summary, LHRH immunization decreased synthesis and storage of LH and decreased storage, but not synthesis of FSH in bulls. The increased synthesis of LH and FSH in nonimmunized, but not LHRH-immunized steers suggests that castration removes the negative feedback on gonadotropin synthesis but that LHRH is still needed for release of these hormones. Androgen replacement with TBA did not restore the negative feedback control of gonadotropin synthesis.

LHRH fusion protein immunization alters testicular development, ultrasonographic and histological appearance of ram testis.

This study was designed to evaluate the effectiveness of a luteinizing hormone releasing hormone (LHRH) fusion protein immunization on reproductive traits in ram lambs including the changes in histology and ultrasonographic appearance of testis. Thirty native ram lambs at 19 weeks of age were divided into control (C, n = 10), immunization (I, n = 10) and castration (E, n = 10) groups. Animals in immunization group were immunized against LHRH using ovalbumin-LHRH-7 (OL) protein generated by recombinant DNA technology as a primary and a booster injection at 19 and 23 weeks of age respectively. Animals were bled via jugular venepuncture at 2-week intervals to determine LHRH antibody and testosterone concentrations. Bi-weekly ultrasonographic examination of the testes was performed to determine the changes in ultrasonographic appearance as the age increased. Biopsied testicular tissues taken at 19, 29 and 41 weeks age were also evaluated. At 41 weeks of age, animals were slaughtered. Semen and epididymis were evaluated for the presence of sperm cells. Testicular development and sperm production were suppressed in the immunized animals. Semineferous tubule diameters decreased, basal membrane of the tubule was thickened and hyalinized in immunized ram lambs. While testes of control animals gained their normal ultrasonographic appearance as the age increased, immunized animals had uniform hypoechogenic testicular structure as observed at 19 weeks of age until slaughter. Simultaneous histological and ultrasonographic evaluations indicated that the changes in testicular histology could partly be monitored via ultrasonographic imaging. Nevertheless, it is difficult to claim that ultrasonographic image reflects the exact changes in such instances. In conclusion, these results indicate that recombinant OL fusion protein is effective in immunocastration in ram lambs and has a potential to be used as an alternative to physical castration. Further research studies should be conducted to help assess reproductive status of testes from ultrasound images.

GnRH or eCG treatment fails to restore reproductive function in GnRH immunized ewes.

This study was designed to evaluate the potential of using eCG or GnRH in restoring reproductive functions in GnRH immunized ewes. Thirty-three multiparous Kivircik ewes were randomly assigned into either control group (n=11) or immunization group (n=22). Ewes were immunized against GnRH by injecting with a cocktail of ovalbumin-LHRH-7 (ovalbumin-GnRH-7) and thioredoxin-LHRH-7 (thioredoxin-GnRH-7) fusion proteins generated by recombinant DNA technology in April. 500 IU eCG or 0.008 mg GnRH analogue was used to induce ovulations. Serum GnRH antibodies were present in animals of the immunized group beginning the second week after the first immunization and maintained throughout the study (14 months). Immunization caused anestrus in immunized ewes. eCG or GnRH analogue administration given after 14 days progestagen (20 mg fluorogestone acetate, FGA) treatment during breeding season (mid July) did not induce ovulation in these ewes. Two more attempts with single or multiple eCG injections failed to induce ovulation in this group as well. It appears that the gonadotropin stimulation was not of adequate time since neither eCG nor GnRH administration was able to restore reproductive function in immunized animals. The immunization effect lasted more than a year. These results suggest that GnRH immunization exerts its effect via the hypothalamo-pituitary axis and that more than such stimulation is required to overcome the reproductive suppression.

Changes in testicular development, ultrasonographic and histological appearance of the testis in buck kids immunized against LHRH using recombinant LHRH fusion protein.

This study was designed to evaluate the effectiveness of recombinant Ovalbumin-LHRL (OL) immunization on changes in testicular size, histological appearance and testosterone production in buck kids. Thirty native buck kids at 18 weeks of age were divided into three groups, control (n = 10), immunization (n = 10) and castration (n = 10) groups. Immunized animals received OL protein generated by recombinant DNA technology. Ultrasonographic and histological examinations of the testes were performed. Animals were slaughtered at 44 weeks of age. Semen and epididymides were evaluated for the presence of sperm cells. Immunized animals generated anti-LHRH antibodies. Testosterone production, testicular and accessory glands development and sperm production were suppressed in the immunized animals (p < 0.01). Semineferous tubule diameters decreased (p < 0.01), basal membrane of the tubule was thickened and hyalinized in immunized kids. Immunization affected ultrasonographic appearance of the testes drastically. While testes of control animals gained their normal ultrasonographic appearance as the age increased, immunized animals had uniform hypoechogenic testicular structure as observed at 18 weeks of age until slaughter. Simultaneous histological and ultrasonographic evaluations indicated that the changes in testicular histology could partly be monitored via ultrasonographic imaging; nevertheless, it is difficult to claim that ultrasonographic image reflects the exact changes in such instances. In conclusion, these results indicate that recombinant OL fusion protein is effective in immunocastration in buck kids and has a potential to be used as an alternative to physical castration. Further researches should be conducted to help assessing reproductive status of testes from ultrasound images.

Luteinizing hormone-releasing hormone fusion protein vaccines block estrous cycle activity in beef heifers.

Two LHRH fusion proteins, thioredoxin and ovalbumin, each containing seven LHRH inserts were tested for their ability to inhibit estrous cycle activity. The objective was to evaluate immune and biological responses from alternating the two fusion proteins in an immunization schedule. One hundred ten heifers were divided equally into 11 groups. Two control groups consisted of either spayed or intact, untreated heifers. Heifers in the other nine groups were immunized on wk 0, 4, and 9. Treatments were immunizations of the same protein throughout or alternating the proteins in different booster sequences. Blood was collected weekly for 22 wk, and serum was assayed for concentrations of progesterone and titers of anti-LHRH. At slaughter, reproductive tracts were removed from each heifer and weighed. Heifers with >or=1 ng/mL of progesterone were considered to have a functional corpus luteum and thus to have estrous cycle activity. All LHRH-immunized groups of heifers had a smaller (P < 0.05) proportion of heifers showing estrous cycle activity after 6 wk than the intact, untreated control group. There was no difference in number of heifers cycling between the immunized groups and the spayed heifers during wk 9 to 22. Anti-LHRH did not differ among immunized groups during wk 1 to 9. Starting at wk 10 and continuing through the conclusion of the study, there was an overall difference among treatment groups for anti-LHRH (P < 0.05). Uterine weights differed among treatments (P < 0.05), with intact control animals having heavier uteri than all other groups (P < 0.05). Uterine weights were negatively correlated with maximum LHRH antibody binding (r = -0.44). In summary, the LHRH fusion proteins were as effective as surgical spaying in suppression of estrous cycle activity, but alternating the two proteins in an immunization schedule did not enhance the immunological or biological effectiveness of the vaccine.

Development of recombinant ovalbumin-luteinizing hormone releasing hormone as a potential sterilization vaccine.

The objective was to develop an immunogenic chimeric ovalbumin-LHRH (ova-LHRH) molecule using genetic engineering. Hybrid ova-LHRH genes with either four or seven LHRH inserts were constructed by cassette mutagenesis and oligonucleotide mismatch mutagenesis. Recombinant ova-LHRH proteins were over-expressed in E. coli strain BL21 (DE3) using a pET expression system, which expresses a target protein with a C-terminal His-Tag. The C-terminal His-Tag allows purification by metal chelation chromatography. The antigenicity and biological effects of these recombinant proteins were tested in mice. In experiment 1, 17 female 7 wk old BALB/c mice were randomly divided into three groups. Six mice were injected with 50 microg of the recombinant ovalbumin (ova) protein. Five mice were injected with 50 microg of the recombinant protein with four LHRH inserts (ova-LHRH-7). Six mice were injected with 50 microg of the recombinant protein with seven LHRH inserts (ova-LHRH-7). One primary immunization using Freund's complete adjuvant was followed by one booster using incomplete adjuvant. Mice were killed 2 wk after the booster, blood collected, and the reproductive tract removed and weighed. Only ova-LHRH-7 decreased (P < 0.01) uterine-ovarian weight (89+/-11 mg) vs control (138+/-6 mg) and ova-LHRH-4 (126+/-16 mg). The genetically engineered molecule with seven LHRH inserts induced LHRH antibody titers which were significantly correlated (r = -0.79) with biological response. In experiment 2, the recombinant ova-LHRH-7 was evaluated at two doses with the adjuvants Zmax and Immumax. Seventy female 6-8 wk old BALB/c mice were randomly divided into seven groups of 10 mice each. Anti-LHRH titers were detected in all of the ova-LHRH-7 immunized mice. Significant decreases were shown in uterine-ovarian weight of the mice by the immunization with 30 microg of ova-LHRH-7 and Zmax (P < 0.005) or 10 microg of ova-LHRH-7 with Immumax (P < 0.025). These data show that the recombinant ova-LHRH-7 protein could have potential as an effective sterilization vaccine.

Expression of biologically active heterodimeric bovine follicle-stimulating hormone in milk of transgenic mice.

Follicle-stimulating hormone (FSH; follitropin) is a pituitary glycoprotein composed of two post-translationally modified subunits, which must properly assemble to be biologically active. FSH has been difficult to purify and to obtain in quantities sufficient for detailed biochemical studies. We have targeted FSH expression to the mammary gland of transgenic mice by using cDNAs encoding the bovine alpha and FSH beta subunits and a modified rat beta-casein gene-based expression system. Lines of bigenic mice expressing both subunits have been generated either by coinjection of the subunit transgenes or by mating mice that acquired and expressed transgenes encoding an individual subunit. Up to 60 international units (15 micrograms) of biologically active FSH per ml was detected in the milk of the bigenic mice. These lines provide a model system for studying the post-transcriptional mechanisms that effect the expression and secretion of this heterodimeric hormone.

Bulls show no preference for a heifer in estrus in preference tests.

The objective of this experiment was to determine whether sexually experienced bulls would demonstrate a preference (using primarily olfaction) between a heifer in estrus and a heifer in diestrus (luteal phase) when physical contact was denied. In Exp. 1, a heifer in estrus and a heifer in diestrus (n = 18 pairs) were individually enclosed in opposite ends of a pen. During each period (n = 18), three bulls were individually introduced into the pen and allowed 5 min to demonstrate preference between the heifer in estrus and the heifer in diestrus. The total time that a bull spent within 2.5 m of either heifer was used to evaluate his preference. The total time that bulls spent adjacent to the heifer in estrus was not greater (P greater than .05) than the total time that bulls spent adjacent to the heifer in diestrus. In Exp. 2, five bulls were used and were evaluated using the same method as in Exp. 1. In addition, the number of flehmen reactions were recorded for each bull. Six heifers were ovariectomized and each heifer was induced into estrus with one of three doses of estradiol 17 beta (5, 10, and 20 mg) over the 5-wk treatment period. Estradiol 17 beta-treated heifers were always paired with a non-estradiol-treated (control) heifer. The goal of Exp. 2 was to determine whether heifers treated with pharmacological doses of estradiol 17 beta would be preferentially selected from non-estradiol-treated (control) heifers by bulls.(ABSTRACT TRUNCATED AT 250 WORDS)

Active immunization of beef heifers against luteinizing hormone: I. Evaluation of protein carriers and adjuvants on antigenicity of LH.

Two experiments were conducted to evaluate two carrier proteins and nine adjuvants in promoting antibody production in heifers immunized against LH. The anti-LH antibody response was evaluated in heifers immunized against LH conjugated to either ovalbumin or keyhole limpet hemocyanin (KLH) (Exp. 1). In Exp. 2, an LH-ovalbumin conjugate was used to evaluate effectiveness of nine different adjuvants in antibody production. Weekly blood samples were collected from all heifers throughout the 23-wk study to determine LH antibody binding activity. In Exp. 1, heifers immunized with the LH-ovalbumin (LH-oval) conjugate had greater LH antibody binding activities (P less than .001) than those immunized with the LH-KLH conjugate. In Exp. 2, nine groups of heifers were immunized with LH-oval suspended in one of nine adjuvants; a 10th group was immunized against ovalbumin alone (control). Only adjuvants that contained at least 40% oil resulted in LH antibody binding activity that differed (P less than .01) from control. These results show that ovalbumin was a superior carrier protein to KLH in enhancing antibody production; adjuvants with greater than 50% oil were superior to those with less oil in promoting LH antibody production.

Active immunization of beef heifers against luteinizing hormone: III. Evaluation of dose and longevity.

Objectives were to evaluate the dose (Exp. 1) and purity of LH preparations (Exp. 2) on the anti-LH antibody response in heifers. Experiment 3 evaluated the longevity of LH immunization on sterility in heifers. In Exp. 1, 115 crossbred heifers were injected every 3 wk for 6 wk with .1, .33, 1.0, 3.0 or 9.0 mg of LH-ovalbumin. Concentrations of anti-LH antibodies generated were quantified by determining the percentage of binding of [125I]LH in serum. Mena LH binding over wk 0 to 12 was greater in heifers immunized with 1.0 mg conjugate than in heifers immunized with other doses (P less than .05). In Exp. 2, LH-ovalbumin conjugates were made from either LH-1, LH-2 or LH-3, which had relative immunological potencies of 2.1, 1.5 and 1.2 x NIH-LH-S1 units/mg, respectively. Forty-eight crossbred beef heifers were immunized against one of these three LH-ovalbumin conjugates, against LH conjugated without ovalbumin (LH-LH), or against ovalbumin alone (Oval). Estrous cycle activity was monitored by measuring serum progesterone concentration. Potency of the LH preparation used in the LH-ovalbumin conjugate was correlated (r = .94) with its ability to produce LH antibodies. In Exp.3, heifers were injected with 1 mg antigen every 2 wk for 10 wk. Five LH-1 heifers and five control heifers were slaughtered for examination of ovaries 10 wk after the last booster injection. The remaining five LH-I and five control animals were placed with a bull 8 wk after the last booster. All five control heifers conceived by 4 +/- 1 wk after placement with the bull whereas the LH-immunized heifers remained acyclic for 42 to 96 wk.

Conception rate, growth, and lactation of dairy heifers treated with recombinant somatotropin.

Eighty Holstein heifers (295 kg; 13 to 16 mo of age) were allotted to two treatments in a completely random design experiment to determine the effect of daily injections of recombinant bovine somatotropin on conception, growth, and subsequent lactation. Heifers were treated with either 41.2 mg of bovine somatotropin or saline daily for 5 mo. Breeding was initiated 2 mo after the start of bovine somatotropin or saline treatment. Conception rates and number of services per conception did not differ between treatments. During the injection period, heifers treated with bovine somatotropin gained .18 kg/d faster than control heifers. During the 5 mo following the treatment period, control heifers gained .12 kg/d faster than the heifers that had received bovine somatotropin so that at the end of this 10-mo period weights of heifers in the 2 groups were similar. The heifers treated with bovine somatotropin had a greater increase in both hip height and pelvic size compared with control heifers during the 5-mo treatment period. Calving difficulty scores were similar between treatments. Treatment with bovine somatotropin prior to and during breeding of these heifers did not affect milk yield after first calving compared with control heifers.

Vaccines against endogenous hormones: a possible future tool in animal production.

Vaccines have been widely used by the livestock industry to control and prevent disease. New technologies now permit development of vaccines against hormones that control reproduction, growth, and lactation in domestic farm animals. Results of research projects studying passive and active immunization against such hormones as estrogen, testosterone, LH, FSH, and somatotropin have demonstrated that reproductive efficiencies and growth can be altered through vaccination. Although immunizations against most of the hormones studied have proven effective in most cases, there remain problems related to longevity, consistency, and efficiency of response both within and between animals. When these areas have been clearly defined for individual hormones, standardized immunization regimens can be developed that will optimize antibody production in the animal, thus providing the animal agriculture industry with a powerful and profitable production tool.

Active immunization of heifers against luteinizing hormone-releasing hormone, human chorionic gonadotropin and bovine luteinizing hormone.

Seventy crossbred heifers were allotted randomly to 10 treatment groups. Treatments consisted of active immunization against ovalbumin (OV) conjugates of luteinizing hormone-releasing hormone (LHRH), human chorionic gonadotropin (hCG) and bovine luteinizing hormone (bLH) with each of three adjuvants. The adjuvants were complete Freund's adjuvant (CFA), M103(6) and 6VR6. Control animals were immunized against OV alone using CFA. Bulls were placed with the heifers following immunization to allow comparison of pregnancy rates between groups. Blood samples were collected weekly for 14 wk to determine antibody concentrations. Significant levels of circulating LH or LHRH antibodies were detected in heifers immunized with each of the hormone conjugates. Complete Freund's adjuvant was the most effective for stimulating antibody response to these antigens; however, M103 was equally effective when used with bLH or hCG conjugates. None of the heifers in the bLH-OV-CFA, bLH-OV-M103 or LHRH-OV-CFA immunization groups was pregnant at slaughter, whereas 71% of the OV-CFA control heifers were pregnant. Fertility suppression may be achieved in the bovine by active immunization against any of these three hormone conjugates. However, the duration of this study (8 wk after immunization) does not allow evaluation of the duration of effectiveness of each of the treatments.