Immunized Boars Research Articles

Structural modeling and mRNA expression of epididymal β-defensins in GnRH immunized boars: A model for secondary hypogonadism in man.

Human secondary hypogonadism is associated with impaired testicular function, however, little is known about its impact on sperm epididymal maturation. Endocrine disruption in the epididymis could impair the secretion of key proteins, such as β-defensins, responsible for spermatozoa maturation during epididymal transit. This study evaluated the sequence and structural similarities between porcine epididymal β-defensins porcine β-defensins (pBD3), pBD4, pBD125, and pEP2C and their human homologs using bioinformatics integrated with information derived from protein databanks. We then verified whether the expression of pBD3, pBD4, pBD125, and pEP2C genes in the testis and epididymis are influenced by disruption of the hypothalamic-pituitary-testicular (HPT) axis in a pig model for male human secondary hypogonadism. Upon modeling porcine β-defensins, structural and functional analysis confirmed the presence of motifs associated with β-defensin function, validating the models generated in silico. pBD3 and pBD4 showed acceptable structural alignments with human β-defensins BDEF103 and BDEF110, respectively. In addition, evaluation of hormonal regulation of β-defensins was assessed by analyzing the expression of these four β-defensins in adult boars immunized against gonadotropin-releasing hormone (GnRH). Our results indicate that HPT axis disruption modifies the expression of pBD3, pBD4, pBD125, and pEP2C in boar testis and epididymis, suggesting an endocrine-dependent regulation of β-defensins in swine epididymis. In conclusion, sequence and structural homology between pBD3 and pBD4 and their human homologs provide a basis for using the pig as a model for the study of human secondary hypogonadism. Further investigation of the human homologs in hypogonadal men could elucidate the connections between fertility and epididymal expression of β-defensins.

Immunization against recombinant GnRH-I alters testicular structure in an experimental boar model

The aim of this study was to evaluate and to compare testicular tissue in immunized and control boars. Eighteen male piglets, aged 12 weeks, were vaccinated twice intramuscularly with a maltose-binding protein-gonadotropin-releasing hormone I hexamer peptide (MBP-GnRH-I6). Blood samples were taken at 12, 18, 21 and 24 weeks of age. Serum concentrations of testosterone and GnRH-I antibodies were determined by radioimmunoassay. The pigs were sacrificed 6 weeks after the second immunization. Testicular weight and size were recorded and tissue samples were collected for histological examination. The results demonstrated that active immunization against MBP-GnRH-I6 increased serum GnRH-I antibody levels (P < 0.05) and reduced serum concentrations of testosterone (P < 0.05) when compared with controls. Histological studies performed on testicular tissue revealed clear signs of atrophy in the MBP-GnRH-I6 immunized pigs, and a significant reduction (P < 0.05) in paired testes weight and size were seen in the treated boars. Microscopically, the mean diameter of the seminiferous tubules was markedly reduced (P < 0.01). Spermatogonia were visible, as well as few spermatocytes, but no spermatozoa were detected in the seminiferous tubules. Ultramicroscopic analysis of testicular tissue revealed an increase in the thickness of the basement membrane and extensive damage in the cell organelles of the treated animals, including small spermatogonial size, decreased number of mitochondria and endoplasmic reticulum in the primary spermatocyte and spermatid, a shallow hollow for nuclear membranes in Sertoli cells and mitochondrial vacuolation in Leydig cells. We conclude that MBP-GnRH-I6 induces severe atrophy in the testes of immunized boars.

The effect of immunization against GnRF on nutrient requirements of male pigs: a review

In most countries, male pigs are physically castrated soon after birth to reduce the risk of boar taint and to avoid behaviours such as fighting and mounting. However, entire male pigs are more feed efficient and deposit less fat than barrows. In addition, many animal welfare organizations are lobbying for a cessation of castration, with a likelihood that this could lead to inferior pork unless an alternative method is used to control boar taint. An alternative to physical castration is immunization against gonadotrophin releasing factor (GnRF) which allows producers to capitalize on the superior feed efficiency and carcass characteristics of boars without the risk of boar taint. From a physiological perspective, immunized pigs are entire males until shortly after the second dose, typically given 4 to 6 weeks before slaughter. Following full immunization, there is a temporary suppression of testicular function and a hormonal status that resembles that of a barrow. Nutrient requirements will be different in these two phases, before and after full immunization. Given that there have been few published studies comparing the lysine requirements of entire males and barrows in contemporary genotypes, it is useful to use gilt requirements as a benchmark. A series of meta-analyses comparing anti-GnRF immunized boars and physical castrates and use of nutritional models suggest that the lysine requirement of entire males before the second immunization is 5% higher than for gilts, from 25 to 50 kg BW, and by 8% from 50 to 95 kg. Given that the penalty in growth performance for having inadequate dietary lysine is greater in males than in gilts or barrows, it is important to ensure that lysine requirements are met to obtain the maximum benefits of entire male production during this phase. After the second immunization, the lysine requirement of immunized males decreases and may become more like that of barrows. In addition, a consistent effect of full immunization is a marked increase in voluntary feed intake from about 10 days after the second dose. Putting these together, the estimated lysine requirement, expressed in terms of diet composition, falls to 94% of the gilt level. Although general principles can be described now, further research is needed to fully define the lysine requirements of immunized boars. It is important that the temporal pattern of tissue deposition rates and feed intake be explored to be incorporated into models to predict nutrient requirements over the period of rapidly changing metabolism.

Active immunization with recombinant GnRH fusion protein in boars reduces both testicular development and mRNA expression levels of GnRH receptor in pituitary

Immunization using recombinant maltose binding protein–gonadotropin releasing hormone (MBP–GnRH6) altered both testicular development and transcription of the pituitary GnRH receptor (GnRHR) gene in boars. Scrotal measurement and blood samples were taken at 4-week interval after immunization at 9 weeks of age. The concentrations of testosterone and anti-GnRH antibodies in serum were determined by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. The results showed that active immunization with MBP–GnRH6 increased the serum concentration of anti-GnRH antibodies ( P < 0.05) and reduced the serum concentration of testosterone ( P < 0.05) as compared with MBP controls. At 25 weeks of age, boars were sacrificed and testes were evaluated histologically. Testicular development was suppressed in the MBP–GnRH6 immunized animals as compared with MBP immunized boars. MBP–GnRH6 immunized pigs exhibited mounting behavior 4 weeks later than MBP immunized boars. No mature spermatozoa were observed from MBP–GnRH6 immunized animals. By real-time quantitative PCR analysis, the amount of GnRHR mRNA in the pituitary tissue was found to be significantly lower in MBP–GnRH6 immunized animals than in controls ( P < 0.05). These data demonstrate that recombinant MBP–GnRH6 was effective in immunological castration in boars.

Individual return to Leydig cell function after GnRH-immunization of boars

Immunization of boars against GnRH inhibits synthesis of testicular anabolic steroids and the sex odour androstenone. Time between second vaccination and return of Leydig cell function is unknown. Six catheterized boars received the second dose (Improvac ®) at 26 weeks of age (day 0). Titre, LH, testicular steroids, and IGF-I were determined in blood until testosterone exceeded 0.5 ng/mL again in all boars. At week 10 the titre was low again. Return of testicular function was preceded by increased pulsatile secretion of LH but onset of steroid synthesis was delayed and highly variable (10–24 weeks) and was not related to the initial antibody titre. A major reason for delayed onset of Leydig cell function apparently is a variable refractoriness against LH.

Short-term endocrine and metabolic reactions before and after second immunization against GnRH in boars

Immunization of boars against GnRH inhibits synthesis of testicular steroids including androstenone (sex odour). Timing of the second vaccination (anamnestic reaction) should occur as late as possible to maintain anabolic effects of testicular hormones, but early enough to remove androstenone from body fat. Five catheterized boars received the second dose (Improvac ®) at age 22 weeks. Titre, hormones and parameters reflecting protein turnover were determined in blood. An increased antibody titre and drop of LH and steroids occurred within 5 days. Metabolism adapted after 7 days. Results from this study in conjunction with previous work suggest that after two doses of Improvac given 4 weeks apart, clearance of androstenone from body fat may be achieved as early as 3 weeks after the second vaccination. Thus, it might be possible to extend the duration of anabolic effect in male pig production.

Effects of Estradiol Infusion in GnRH Immunized Boars on Spermatogenesis

Active immunization of boars against gonadotropin-releasing hormone (GnRH) inhibits luteinizing hormone (LH) and testicular steroids, so that mitosis of spermatogonia is reduced and apoptosis increased. To clarify whether high amounts of estrogens which are synthesized in the boar testis support spermatogenesis, a group of 6 boars was immunized against GnRH and then infused for 7 weeks with estradiol (E2-17beta). For comparison, intact boars and immunized boars were infused with saline only. Testicular tissue was then analyzed by immunocytochemistry for apoptosis (TUNEL, EM), mitosis (Ki67), and estrogen receptor alpha (ER alpha). The specificity of ER alpha staining was confirmed by RT-PCR and Western blot. Immunization decreased LH and testosterone to minimal concentrations in immunized and E(2)17beta-infused immunized boars, whereas follicle-stimulating hormone (FSH) was not significantly altered. Estradiol decreased to base levels after immunization. Infusion increased E2-17beta in peripheral blood plasma of the immunized boars to physiological levels. Except for A-spermatogonia, all spermatogenic cells decreased after immunization by about 60%. After estradiol infusion, cell counts increased again and were intermediate between control and immunized boars. Mitosis of spermatogonia was reduced by nearly 50% due to immunization but was partly restored by E2-17beta infusion. Expression of ER alpha was localized in spermatogonia, suggesting stimulation of mitosis, which was further confirmed due to its predominant occurrence in stage I of the seminiferous epithelial cycle (main stage of cell division). Apoptosis was minimal in boars but elevated in the other 2 groups. Data showed that estrogens in physiological concentrations supported mitosis but were not sufficient to normalize sperm production because apoptosis was still high.

Characterization of 11β-Hydroxysteroid Dehydrogenase Activity in Testicular Tissue of Control and GnRH-Immunized Boars as a Possible Regulator of Spermatogenesis

Glucocorticoids are involved in the regulation of spermatogenesis in the boar testis by initiating apoptosis in early stages of germ cell development. Because cortisol activity is modulated by the 11beta-hydroxysteroid dehydrogenase system (11beta-HSD), the present study determined both 11beta-activating (reductive) and inactivating (oxidative) enzyme activities in testicular tissue preparations of control boars (n = 5), GnRH-immunized boars (n = 5), and immunized, estradiol-infused boars (n = 6) by radioenzyme assay based on the conversion of tritiated cortisol to cortisone in the presence of NAD+ (inactivation) or tritiated cortisone to cortisol in the presence of NADPH (activation). The presence of both isoforms, 11beta-HSD 1 and 11beta-HSD 2, was confirmed by RTPCR in testicular tissue of control boars. Additionally, cortisol, testosterone, estradiol, and LH were determined in blood plasma sampled twice before killing. Immunization led to a drop of LH from 4.3 +/- 0.3 pmol/L to 1.0 +/- 0.3 pmol/L (testosterone: 11.63 +/- 0.83 nmol/L vs. 0.28 +/- 0.07 nmol/L; 17beta-estradiol: 512.61 +/- 47.60 pmol/L vs. 77.05 +/- 14.00 pmol/L). Low 11beta-HSD reductive activity was found in boars (1 pmol steroid x min (-1) x mg (-1)). It decreased to trace amounts in immunized boars (0.07 pmol steroid x min (-1) x mg (-1)). Oxidative activity was found in boars with 10.19 +/- 2.28 pmol steroid x min (-1) x mg (-1) protein. Immunization led to a sharp decrease (0.08 +/- 0.03 pmol x min (-1) x mg (-1)). Infusion of 17beta-estradiol significantly elevated peripheral estradiol concentrations to 752.07 +/- 24.19 pmol/L which still is a physiological concentration in this species. The infusion led to a minimal reductive activity (0.04 pmol steroid x min (-1) x mg (-1)), but led to a 6-fold rise of the 11beta-HSD oxidative activity to 0.47 +/- 0.14 pmol x min (-1) x mg (-1) compared to immunized boars. It is concluded that the 11beta-HSD system is involved in the regulation of cortisol activity in the testis and thus in the regulation of spermatogenesis.

Involvement of glucocorticoids in testicular involution after active immunization of boars against GnRH.

Active GnRH immunization of boars inhibits LH and testicular steroids but the consequences for spermatogenesis are unknown. Six boars were immunized three times against GnRH at 20, 24 and 28 weeks. Another six boars served as controls. Plasma LH and FSH were determined at 28 and 31 weeks. Testosterone and cortisol were determined before killing the pigs at 32 weeks. Tissue samples were taken for histology and fluid from the seminiferous tubuli for steroid determination. Individual germ cells were counted in histological sections. The glucocorticoid receptor (GCR), mitosis of spermatogonia and apoptosis were characterized by immunocytochemistry. Immunization reduced LH and testosterone to base levels whereas FSH was not changed. Testis weight was reduced by 64% due to a loss of Leydig cell cytoplasm (90.3%) and a decrease of tubule diameters (60.6%). Except for A-spermatogonia, all other spermatogenic cells were reduced by about 60%. Mitosis was reduced in immunized boars. Expression of GCRs was limited to spermatogonia and differed between immunized boars (8% of spermatogonia) and controls (2%). In the controls, androgen concentrations in tubular fluid were tenfold higher compared with immunized boars. Cortisol concentrations were of the order of 40 nmol/l both in the tubular fluid and blood plasma. These concentrations did not differ between groups. Apoptosis occurred only in spermatogonia and pachytene spermatocytes and was twofold higher in immunized boars compared with controls. Thus the availability of glucocorticoids in the tubuli and the expression of GCRs initiate apoptosis, which in turn reduces sperm yield. Testosterone is known to be an inhibitor of GCR expression, thus increasing the efficiency of spermatogenesis.

Active immunization of boars against GnRH does not affect growth hormone but lowers IGF-I in plasma

Active immunization of boars against GnRH inhibits the endocrine testicular function and thus the occurrence of boar taint at slaughter. Some studies indicate that at least a part of the boar-specific growth potential is still retained. To clarify possible mechanisms, key hormones which are involved in growth regulation were measured in four control boars (C), five immunized boars (I) and three barrows (B). Immunization started at an age of 10 weeks and was repeated twice. Growth hormone (GH), IGF-I, LH, estradiol and glucocorticoids were measured radioimmunologically in blood plasma (daily samples or windows) which had been obtained via permanent catheters. GH was in a similar order both for C and I (4.19 and 3.88 ng/ml; n.s.) but low in B (2.88 ng/ml). LH was minimal in I (130 pg/ml), highest in B (550 pg/ml), and intermediate in C (220 pg/ml). IGF-I was significantly higher in C compared to both, I and B (239 vs. 123 ng/ml; P≤0.001). Estradiol was decreased in I compared to C (24.11 vs. 90.21 pg/ml, P≤0.001) but glucocorticoids did not differ between C (17.53 ng/ml) and I (17.51 ng/ml). It is concluded that ‘immunological castration’ allows the postnatal imprinting of a male-specific level of GH secretion but no concomitant rise of IGF-I due to the lack of estrogens which couple GH and IGF-I. The remaining anabolic potential thus is due to a late onset of testicular inhibition in some immunization trials.

Active immunization of boars against GnRH at an early age: consequences for testicular function, boar taint accumulation and N-retention

A total of ten boars were immunized with a vaccine against GnRH and the parameters for testicular function and N-retention compared with eight intact boars and eight barrows. The immunization of boars occurred at 10, 16 and 23 weeks of age. In six pigs from each group the nitrogen retention was determined at 20/21 weeks. Blood for testosterone determination was collected daily and fat biopsies for androstenone measurement every 2nd week from week 17 to 26 in all boars. After slaughter, testes size, backfat thickness and skatole concentrations in fat were determined. The immunized boars revealed significant lower ( P≤0.001) testes weights (100.5 vs. 882.6 g), plasma testosterone concentrations (0.11 vs. 2.77 ng/ml) and fat androstenone concentrations (0.06 vs. 1.31 μg/g, week 26) compared to controls. Skatole concentrations also differed significantly between immunized boars and controls (14.4 vs. 109.2 ng/g fat). The immunized boars had a significantly poorer feed conversion rate (3.82 vs. 3.01 kg/kg) and nitrogen retention (22.97 vs. 31.24 g/day) compared to boars. Barrows did not differ significantly from the immunized boars. It is concluded that an early and repeated immunization against GnRH is highly effective for a complete androstenone suppression and a considerable reduction of skatole but it also abolishes the boar-specific anabolic potential.

Has active immunization against gonadotrophin-releasing hormone any effect on testis innervation in the pig? An immunohistochemical study.

The innervation of porcine testes was studied in intact animals and in boars undergoing active immunization against gonadotrophin-releasing hormone (GnRH) by means of immunohistochemistry using antibodies to tyrosine hydroxylase (TH), dopamine beta-hydroxylase (D beta H), vasoactive intestinal polypolypeptide (VIP), neuropeptide Y (NPY), synaptosome-associated protein of 25 kDa (SNAP-25) and protein gene product 9.5 (PGP 9.5). Moreover, the distribution of luteinizing hormone (LH) receptors in clusters of Leydig cells was also investigated. To identify these cells easily, either the NADPH-diaphorase histochemical technique or the Mayer counter-staining procedure was applied. Differences in the distribution pattern and relative density of particular subsets of intratesticular nerve fibres were observed in immunized boars as compared to those found in the intact animals. In the testes of non-treated animals, only single TH-immunoreactive (TH-IR) nerve fibres were observed. However, many D beta H-IR nerve terminals surrounded blood vessels in the tunica albuginea and parenchyma. Very scarce VIP-IR nerves occurred only in the tunica albuginea, mainly in close vicinity to blood vessels. Immunoreactivity to NPY occurred in single nerve fibres. Immunoreactivity to SNAP-25 and PGP 9.5 was found in single nerve fibres distributed mainly in the tunica albuginea. The interstitial cells were heavily stained for LH-receptors and NADPH-diaphorase. In the testes of immunized animals, only single TH-IR nerve fibres, scattered mainly in the tunica albuginea, were observed. Some TH-IR nerve terminals were also encountered in the parenchyma of the organ, where they were always associated with blood vessels. D beta H-IR nerve fibres formed a dense network distributed throughout the testis in association with the capsule, vasculature and interstitium. Some fibres were observed to run between seminiferous tubules. VIP-IR nerve fibres were located in the neighbourhood of blood vessels in the tunica albuginea and parenchyma. Only single VIP-IR nerves were found between seminiferous tubules. Numerous NPY-IR nerve fibres occurred in the tunica albuginea and parenchyma of the organ. SNAP-25-IR and PGP 9.5-IR nerve terminals formed a dense network distributed throughout the testis and many fibres were observed between seminiferous tubules. Interstitial cells were very weakly stained for LH receptors or NADPH-diaphorase.

Active immunization of boars against gonadotropin releasing hormone. II. Effects on libido and response to testosterone propionate

Of 20 sexually mature Duroc boars showing normal libido, 10 were actively immunized against gonadotropin releasing hormone (GnRH). After immunization against GnRH, boars showed minimal sexual interest in an estrous female, while untreated boars showed normal libido. Eight of the boars actively immunized against GnRH were randomly assigned to treatment (T) or control (C) groups. Boars in the T and C groups were given testosterone propionate or vehicle, respectively, on Days 0, 5, 10, and 15. Boars in both groups were observed for libido in the presence of an estrous female every 4 d for 28 d. Mean libido score for T boars increased gradually until all boars displayed maximum libido on Day 20, but libido returned to low levels on Day 28. In contrast, C boars remained sexually inactive throughout the study. The results of this study indicate that active immunization of sexually mature boars eliminates sexual behavior and that sexual behavior can be restored quickly by administering testosterone propionate.

A selective immunization procedure against 5α-androstenone in boars

ABSTRACTAn auto-immunization procedure selective for boar 5α-androstenone is described. Immunization of boars with a conjugate of androstenone and bovine serum albumin as a carrier protein significantly reduced the accumulation of androstenone in adipose tissue from a mean value of 0·77 (s.e. 0·19)mg/kg fat for untreated boars to 0·24 (s.e. 0·06) mg/kg fat for treated animals. No detrimental side-effects were observed in the immunized animals, and the advantages of male-type growth and carcass characteristics were preserved.