The objective of this research was to examine the population structure of full-blood (100%) Wagyu cattle registered in the United States with the American Wagyu Association, with the aim of estimating and comparing the levels of inbreeding from both pedigree and genotypic data. A total of 4132 full-blood Wagyu cattle pedigrees were assessed and used to compute the inbreeding coefficients (F-IT and F-ST) and the effective population size (N-e) from pedigree data for the period 1994 to 2011. In addition to pedigree analysis, 47 full-blood Wagyu cattle representing eight prominent sire lines in the American Wagyu cattle population were genotyped using the Illumina BovineSNP50 BeadChip. Genotypic data were then used to estimate genomic inbreeding coefficients (F-ROH) by calculating runs of homozygosity. The mean inbreeding coefficient based on the pedigree data was estimated at 4.80%. The effective population size averaged 17 between the years 1994 and 2011 with an increase of 42.9 in 2000 and a drop of 1.8 in 2011. Examination of the runs of homozygosity revealed that the 47 Wagyu cattle from the eight prominent sire lines had a mean genomic inbreeding coefficient (F-ROH) estimated at 9.08% compared to a mean inbreeding coefficient based on pedigree data of 4.8%. These data suggest that the mean genotype inbreeding coefficient of full-blood Wagyu cattle exceeds the inbreeding coefficient identified by pedigree. Inbreeding has increased slowly at a rate of 0.03% per year over the past 17years. Wagyu breeders should continue to utilize many sires from divergent lines and consider outcrossing to other breeds to enhance genetic diversity and minimize the adverse effects of inbreeding in Wagyu.

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 β-subunit and common α-subunit genes (P<0.001). Castration increased expression of LH β-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.

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.

Immunizing farm animals against luteinizing releasing hormone (LHRH) is an alternative to castration (Thompson, 2000). It suppressed testicular development and reduced sexual activities in domestic species (Adams and Adams, 1992). It has been reported that immunizing male animals against LHRH had no negative effect on carcass quality (Kiyma, et al., 2000 and Huxol et al., 1998). A recombinant fusion protein, ovalbumin-LHRH-7 was developed (Zhang et al., 1999). The immunocastrate effect of this recombinant protein was demonstrated in farm animals (Ulker et al., 2003).The aim of the present study was to compare the carcass traits of goat kids intact, surgically castrated and actively immunized against LHRH using ovalbumin-LHRH-7 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.

J. Anim. Sci. Vol. 86, E-Suppl. 2/J. Dairy Sci. Vol. 91, E-Suppl. 1 W150 Immunosterilization of bitches with an anti-LHRH vaccine using CpG ODN as an adjuvant. R. Zanella1, M. Ragagnin de Lima*4, J. J. Reeves1, V. Conforti2, D. DeAvila1, A Ferreira Marques4, S. A. Messina1, R Bogden3, and E. L. Zanella4, 1Washington State University, Pullman, 2Cincinnati Zoo & Botanical Garden, Cincinnati, OH, 3Amplicon Express, Pullman, WA, 4Universidade de Passo Fundo, Passo Fundo, RS, Brazil.

Objectives of this study were (1) to determine if trenbolone acetate (TBA) co-administered with LHRH immunization would suppress reproductive function in beef bulls and (2) to examine the effects of LHRH immunization and TBA treatment on pituitary function. To address these objectives 44 Angus x Hereford bull calves (mean BW = 225 ± 2 kg; mean age = 187 ± 6 d) were randomized into eight treatments in a 2 × 2 × 2 factorial experiment, with castration, LHRH immunization, and TBA administration as treatment factors. Calves immunized against LHRH received a primary injection of ovalbuminLHRH-7 fusion protein on d 0, followed by two booster injections on d 42 and 196. Calves treated with TBA were implanted on d 224. Mean LHRH antibody binding activity in serum increased after each booster for immunized calves, but was negligible in non-immunized animals throughout the experiment. Concentrations of testosterone in serum were lower (P < 0.0001) by d 84 and scrotal circumference smaller (P < 0.05) by d 168 in LHRH immunized bulls compared to non-immunized bulls. Treatment with TBA tended (P = 0.07) to decrease concentrations of testosterone in serum from bulls. Testes + epididymides weights at slaughter (d 272) were lighter (P < 0.0001) for immunized compared to non-immunized bulls. Both LHRH immunization and castration resulted in decreased anterior pituitary stores of LH and FSH (P < 0.001). Immunization against LHRH suppressed expression of the LH β, and common α-subunit genes (P < 0.0001), while castration increased expression of the same two genes (P = 0.02). Synthesis and storage of LH and FSH, as measured by pituitary LH and FSH content and expression of the LH β-subunit and common α-subunit genes, was suppressed by LHRH immunization.

The first objective of this study was to evaluate the effectiveness of an LHRH fusion protein in suppressing reproductive function in male Sprague Dawley rats (n=90). Even though this procedure is producing an autoimmune disease, we hypothesize that immunocastration would not have a negative impact on the lifespan of rats. To test this hypothesis male rats were immunized with a recombinant ovalbumin-luteinizing hormone-releasing hormone (oval-LHRH) fusion protein to examine the effects of immuno-castration on lifespan. The immunized rats were given a primary injection of oval-LHRH with complete Freund's adjuvant followed by two boosters of oval-LHRH and incomplete Freund's 4 and 8 wk later. The intact rats in the control group were injected with recombinant ovalbumin alone using the same adjuvant. Blood samples and testicular measurements were collected on 28-day intervals. Efficacy of the vaccine was determined by concentrations of LHRH antibodies associated with testicular atrophy. Of the LHRH immunized rats 97.8% responded generating antibodies against LHRH coupled with testicular size reduction. The effect of LHRH immunization on lifespan was examined by comparing the age at which time 50% of each treatment group died. This 50% survival time for the intact control rats was 22.5 with a SE of 1.5 and the immunized 26 months with a SE of 0.5. Based on the Wilcoxon test for comparison of survival curves, there was a notable, but non-significant difference in survival between immunized and control groups (P=0.074). This is an important observation in the consideration of using such vaccines in controlling reproduction in wild, feral or pet animals.

This study was carried out to evaluate the effect of a luteinizing hormone-releasing hormone (LHRH) fusion protein vaccine vs. surgical castration on the chemical composition and fatty acid profile of beef cattle (Bos indicus) slaughtered at three years of age. Thirty bulls (Nellore-cross) were divided into three groups: immunized against LHRH fusion protein, castrated or left intact (control). These animals were 24 months old at the initiation of the study and ranged on Brachiaria grass in Mato Grosso, Brazil. Meat of intact bulls presented a lower fat content compared to castrated or surgically castrated animals. For the immunized and surgically castrated animals there were differences in fat and cholesterol concentrations. Although the fatty acid profiles of the three treatments were similar in composition, there were differences in the concentrations of some fatty acids (miristic, C14:0; stearic, C18:0; linolenic, C18:3n-3; and eicosapentaenoic, C22:5n-3). Meat samples of surgically castrated animals had less polyunsaturated (PUFA) and monounsaturated (MUFA) fatty acids (8.2 and 37.7%, respectively), higher saturated (SFA) fatty acids (52.2%) and a lower PUFA/SFA ratio (0.16) and were different compared to intact and immunized bulls. The PUFA/SFA ratio increased as the fat content decreased. On the other hand, surgically castrated bulls had a higher lipid and lower cholesterol contents. It is concluded that immunization against LHRH using new recombinant LHRH fusion proteins could be an alternative to physical castration to improve carcass quality in cattle production.

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.

Sixteen native ram lambs weaned at 10 wk of age were divided into two groups. Eight animals were immunized against LHRH with a mixture of two fusion proteins: ovalbumin-LHRH-7 and thioredoxin-LHRH-7. The immunized lambs received a primary immunization plus two booster immunizations at 4 and 12 wks. Animals in the control group (n=8) were not treated. Scrotal measurements and blood samples were taken at 2-week intervals. Beginning at 25 wk of age, semen was collected and sexual behaviour was evaluated on a weekly basis. At 35 and 37 wk of age testes and accessory glands of all animals were subjected to ultrasound scanning. At 37 wk of age animals were slaughtered and testes were evaluated histologically. Serum LHRH antibodies (P<0.01) were detected in animals of the immunized group which had reduced serum testosterone concentrations (P<0.01). Testicular development was suppressed in the immunized animals (P<0.01). Immunized animals exhibited mounting activity 5 wks later than control animals. No mature spermatozoa containing ejaculates were collected from immunized animals. Control animals had moderately echogenic ultrasonographic appearance at 37 wk age, whereas immunized animals had hypoechogenic images. Mean seminiferous tubule diameter in immunized lambs was significantly smaller than that in control lambs. Basal membrane was thickened and hyalinized; there was an increase in peritubular connective tissue. No proliferating spermatogonia or mature spermatozoa were present in the tubules in these animals. There were no differences in the ultrasonographic appearance of prostate and vesicular gland between control and immunized animals. The LHRH recombinant fusion proteins were effective in immunological castration in ram lambs when started at 10 wk of age as noted by differences in serum testosterone, testicular histology and ultrasonographic appearance of testis and weight of accessory sex glands. Determining the effects of immunization on ultrasonographic appearance of the testis related to time after immunization requires further investigations.

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.

Mitochondrial transcription factor A (TFAM), a nucleus-encoded protein, regulates the initiation of transcription and replication of mitochondrial DNA (mtDNA). Decreased expression of nuclear-encoded mitochondrial genes has been associated with onset of obesity in mice. Therefore, we hypothesized genetic variants in TFAM gene influence mitochondrial biogenesis consequently affecting body fat deposition and energy metabolism. In the present study, both cDNA (2259 bp) and genomic DNA (16,666 bp) sequences were generated for the bovine TFAM gene using a combination of in silico cloning with targeted region PCR amplification. Alignment of both cDNA and genomic sequences led to the determination of genomic organization and characterization of the promoter region of the bovine TFAM gene. Two closely linked A/C and C/T single nucleotide polymorphisms (SNPs) were found in the bovine TFAM promoter and then genotyped on 237 Wagyu x Limousin F2 animals with recorded phenotypes for marbling and subcutaneous fat depth (SFD). Statistical analysis demonstrated that both SNPs and their haplotypes were associated with marbling (P = 0.0153 for A/C, P = 0.0026 for C/T, and P = 0.0004 for haplotype) and SFD (P = 0.0200 for A/C, P = 0.0039 for C/T, and P = 0.0029 for haplotype), respectively. A search for transcriptional regulatory elements using MatInspector indicated that both SNPs lead to a gain/loss of six putative-binding sites for transcription factors relevant to fat deposition and energy metabolism. Our results suggest for the first time that TFAM gene plays an important role in lipid metabolism and may be a strong candidate gene for obesity in mammals.

265 with bulls surgically castrated on d 14 gaining the least (0.50 kg). Steers had a tendency (P = 0.06) to have higher total ADG when compared to calves that arrived as bulls. Neither method nor timing of castration affected the number of bulls treated for respiratory disease, the number of antibiotic treatments required/calf, or medication costs/calf (P ≥ 0.54). However, calves that arrived as steers had a lower percentage of calves that received the first and second round of antibiotic treatments, number of antibiotic treatments required/calf, and medication costs when compared to bulls (P < 0.04). This study indicated that method and timing of castration would impact overall growth performance of newly arrived stocker-cattle; however, if calves are castrated prior to arrival, ADG and morbidity could be enhanced.

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.