The distribution and chemical coding of urinary bladder trigone-projecting neurons in testicular and aorticorenal ganglia in male pigs.

Combined retrograde tracing and double-labelling immunofluorescence were used to investigate the distribution and chemical coding of neurons in testicular (TG) and aorticoerenal (ARG) ganglia supplying the urinary bladder trigone (UBT) in juvenile male pigs (n=4, 12 kg. of body weight). Retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the bladder trigone under pentobarbital anesthesia. After three weeks all the pigs were deeply anesthetized and transcardially perfused with 4% buffered paraformaldehyde. TG and ARG, were collected and processed for double-labelling immunofluorescence. The expression of tyrosine hydroxylase (TH) or dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), nitric oxide synthase (NOS) and vesicular acetylcholine transporter (VAChT) were investigated. The cryostat sections were examined with a Zeiss LSM 710 confocal microscope equipped with adequate filter blocks. The TG and ARG were found to contain many FB-positive neurons projecting to the UBT (UBT-PN). The UBT-PN were distributed in both TG and ARG. The majority of them were found in the right ganglia, mostly in TG. Immunohistochemistry disclosed that the vast majority of UBT-PN were noradrenergic (TH- and/or DBH-positive). Many noradrenergic neurons contained also immunoreactivity to NPY, SOM or GAL. Most of the UBT-PN were supplied with VAChT-, or NOS- IR (immunoreactive) varicose nerve fibres. This study has revealed a relatively large population of differently coded prevertebral neurons projecting to the porcine urinary bladder. As judged from their neurochemical organization these nerve cells constitute an important element of the complex neuro-endocrine system involved in the regulation of the porcine urogenital organ function.

Stability of αB-crystallin gene expression in canine mammary gland neoplasms. Should it be considered as circulating tumor cell genetic marker?

αB-crystallin is a member of a small family of thermal shock proteins that protects cells from stress. Because of lack of its expression in peripheral blood leukocytes, it was proposed as a molecular marker of circulating tumor cells in canine mammary gland tumors. The aim of the present study was to determine if αB-crystallin shows stability of expression, what is the requirement for this type of marker. It was also assessed whether there is co-expression of αB-crystallin with the basal marker, cytokeratin 17. For this purpose, samples of various types of canine mammary gland tumors of epithelial origin, were selected. Using RT-qPCR, we have found αB-crystallin and cytokeratin 17 co-expression in benign and malignant canine mammary gland tumors. It has been demonstrated that the expression of αB-crystallin in tested neoplastic samples is not stable in comparison to the control group. Furthermore αB-crystallin overor down- expression was associated witch the same cytokeratin 17 pattern. αB-crystallin can be a marker of circulating tumor cells in the bloodstream, but for cancers in which basal marker expression occurs and thus not universal for all cancers originating from the mammary gland tissue.

Immunohistochemical Characterization of Sympathetic Chain Ganglia (SChG) Neurons Supplying the Porcine mammary Gland.

The aim of this study was to investigate the chemical coding of mammary gland-projecting SChG neurons using double-labelling immunohistochemistry. Earlier observation showed that after injection of the retrograde tracer fast blue (FB) into the second, right thoracic mamma, FB+ mammary gland-projecting neurons were found in Th1-3, Th9-14 and L1-4 right SChG. The greatest number of FB+ nerve cell bodies was observed in Th10 (approx. 843) and Th11 (approx. 567). Neurons projecting to the last right abdominal mamma were found in L1-4 SChG. The greatest number of FB+ neurons was observed in L2 (approx. 1200). Immunohistochemistry revealed that the vast majority of FB+ mammary-projecting neurons contained immunoreactivities to TH (96.97%) and/or DßH (95.92%). Many TH/DßH-positive neurons stained for SOM (41.5%) or NPY (33.2%), and less numerous nerve cells expressed VIP (16.9%). This observation strongly corresponds to the results of previous studies concerning the immunohistochemical characterization of nerve fibres supplying the porcine mammary gland.

Expression of VPAC1 receptor at the level of mRNA and protein in the porcine female reproductive system.

The presence and distribution of vasoactive intestinal polypeptide (VIP) receptor VPAC1 was studied in the ovary, oviduct and uterus (uterine horn and cervix) of the domestic pig using methods of molecular biology (RT-PCR and immunoblot) and immunohistochemistry. The expression of VPAC1 receptor at mRNA level was confirmed with RT-PCR in all the studied parts of the porcine female reproductive system by the presence of 525 bp PCR product and at the level of proteins by the detection of 46 kDa protein band in immunoblot. Immunohistochemical stainings revealed the cellular distribution of VPAC1 receptor protein. In the ovary it was present in the wall of arterial blood vessels, as well as in the ovarian follicles of different stages. In the tubular organs the VPAC1 receptor immunohistochemical stainings were observed in the wall of the arterial blood vessels, in the muscular membrane, as well as in the mucosal epithelium. The study confirmed the presence of VPAC1 receptor in the tissues of the porcine female reproductive tract what clearly shows the possibility of influence of VIP on the porcine ovary, oviduct and uterus.

The expression of mitochondrial, cytoplasmic and extracellular superoxide dismutase in the colonic wall of pigs suffering from swine dysenteria.

The expression of 3 types of peroxide dismutase (SOD1, SOD2 and SOD3) was studied with Real-Time PCR in the colonic wall of domestic pig suffering from swine dysentery. The expression of enzymes was studied separately in the mucosa and the muscular membrane. It was found that in the mucosa the expression of SOD1 (cytoplasmic) did not change, while the levels of expression of mitochondrial SOD2 and extracellular SOD3 were raised in inflamed colon. More dramatic changes were seen in the muscular mebrane where expression of SOD1 rose twice, this of SOD2 rose ca. 5-fold and the expression of SOD3 rose dramatically, even 30-fold. The obtained data are contradictory to findings in other types of colonic inflammation, which were studied either in the whole colonic wall, or in mucosa alone. The results show a very strong reaction of antioxidant systems in the muscular membrane in the enteritis.

The application of circulating tumor cells detecting methods in veterinary oncology.

Cancers are one of the most common diseases affecting dogs. Many of them develop spontaneously and their biology and histopathology shows many similarities to human cancers. What more, it is proved that there are much more analogies in molecular mechanisms of cancer development between these two species. Human oncology is seeking more and more efficient methods for an early disease detection which results directly in the extended life expectancy of patients affected. One of the most modern trends in the diagnosis of cancer is to detect circulating tumor cells (CTC) in the blood of patients. It is known that these cells are responsible for the formation of metastases in distant organs what results in the patient death. Moreover, it's confirmed that CTC are already present in patients' bloodstream in the early stages of tumor development. There is no doubt that mechanism of metastasis development in dogs is identical and thus the CTC are also present in their bloodstream. Despite the intense researches there is still no optimal method of isolating cancer cells from the blood where they occur extremely rarely. The purpose of this study is to analyze the implications of the detection methods of tumor cells in the blood in veterinary oncology.

Nitric oxide in the bovine oviduct: influence on contractile activity and nitric oxide synthase isoforms localization.

The oviducts of 64 Holstein cows in luteal (early I, early II and late) and follicular phases were evaluated to determine the protein expression and mRNA transcription of different nitric oxide synthase isoforms (eNOS, iNOS, nNOS) as well as the effect of nitric oxide (NO) on spontaneous contractility in vitro. The expression patterns of nitric oxide synthase (NOS) isoforms in isthmus and ampulla (n = 6 for each phase) were determined by immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. In the contractility studies, longitudinal and circular isolated strips of isthmus and ampulla (n = 10 for each phase) of oviducts located ipsilateral to the luteal structure or preovulatory follicle were treated as follows: a) L-arginine, an endogenous NO donor (10(-8) to 10(-3)m), b) N(ω)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor (10(-5)m) and L-arginine (10(-3)m), c) methylene blue (MB), an inhibitor of soluble guanylate (10(-5)m) and L-arginine (10(-3)m) and d) sodium nitroprusside (SNP), an exogenous NO donor (10(-8) to 10(-4)m). Immunohistochemical evaluation revealed that endothelial NOS (eNOS) expression detected in epithelial layer of isthmus and ampulla was strong in early I luteal phase, moderate in follicular phase and weak in other phases. Neuronal NOS (nNOS) immunoreactivity was strong in isthmus and moderate in ampulla, and staining of nerve fibers was observed mostly in early I luteal and follicular phases. All eNOS, nNOS and inducible NOS (iNOS) isoforms were detected by RT-PCR. eNOS and iNOS proteins were evident, whereas nNOS was undetectable by Western blot analysis in the tissue examined. L-arginine applied alone or after L-NAME did not alter or increase the contractile tension of the strips in most tissues examined. However, L-arginine applied after MB increased contractile tension in the strips of ampulla and longitudinal isthmus from early I luteal phase and circular isthmus from follicular phase but decreased it in isthmus from early II luteal phase. SNP differentially modulated oviductal contraction depending on the type of muscular strips and period examined. These results showed the estrous phase-dependent changes related to endogenous NO system which might be of physiological importance to the oviduct for secretory and ciliary functions involved in gametes and embryo(s) transportation.

The influence of ileitis on the neurochemistry of the caudal mesenteric ganglion in the pig.

BACKGROUND: Some literature data suggest that there is a regulatory neuronal circuit between the small and the large bowel. To verify this hypothesis the present study investigated: (i) the distribution, chemical coding and routing of caudal mesenteric ganglion (CaMG) neurons participating in an intestinointestinal reflex pathway involving ileal descending neurons and viscerofugal colonic neurons and (ii) possible changes in the neuroarchitecture of this pathway evoked by chemically induced ileitis in juvenile pigs (n=16). METHODS: Combined retrograde tract tracing and transections of the intermesenteric or caudal colonic nerves were applied. In addition, double immunostainings was used to investigate the chemical coding of retrogradely labeled CaMG neurons and intraganglionic nerve terminals apposed to them, under normal and inflammatory conditions. KEY RESULTS: The majority of the ileum-projecting neurons were found in the caudal part of CaMG. Disruption of particular nerve pathways resulted in diminished number of retrogradely labeled neurons, ipsilateral to the side of manipulation. In normal pigs, ileum-projecting CaMG neurons stained for tyrosine hydroxylase, dopamine-ß-hydroxylase, neuropeptide Y (NPY), somatostatin and galanin (GAL). The number and chemical coding of the neurons in the inflamed animals were similar to those observed in the normal pigs. However, in the inflamed pigs, the number of NPY-, GAL- or substance P-positive nerve terminals supplying retrogradely labeled neurons was increased. CONCLUSIONS & INFERENCES: The present results suggest that inflammatory processes of the porcine ileum are able to induce changes in the intraganglionic architecture of a sympathetic ganglion located at discrete distance from the affected bowel segment.

Analysis of the chemical coding of neurons in the intermediate thoracic ganglion of the pig.

The pig has been widely used as a model in cardiovascular research. A unique feature of the porcine extrinsic sympathetic cardiac nerves is that they arise from intermediate ganglia in the thoracic cavity. The localization and pattern of distribution of nerve cell bodies and fibers containing tyrosine hydroxylase (TH), dopamine B-hydroxylase (DBH), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), methionine-enkephalin (MET) as well as calcitonin gene-related peptide (CGRP), substance P (SP) and pituitary adenylate cyclase-activating peptide (PACAP) was studied with immunohistochemistry. Almost all the neurons showed immunoreactivity to TH. Immunoreactivity to NPY, VIP, SOM, GAL, MET and PACAP was displayed by nerve cell bodies while nerve fibers exhibited immunoreactivity to all the neuropeptides studied. Therefore, it seems that the chemical coding of neurons and especially nerve fibers in the porcine intermediate ganglion share general similarities (with certain neurochemical variability), with porcine prevertebral ganglia (e.g., celiacomesenteric and caudal mesenteric ganglia).

Partial sequence analysis of the L1 gene of bovine papillomavirus type 1 detected by PCR with MY09/MY11 primers in equine sarcoids in Poland.

BPV-1 is now recognized as a main etiological agent of equine sarcoids. The etiopathogenesis of the equine sarcoids is equivocal and is not yet fully understood. The aim of the present study was to analyse a partial sequence of the L1 gene of BPV associated with equine sarcoids in Polish horses. After clinical diagnosis, 40 skin lesions obtained from 29 horses were collected. The amplicons of a fragment of BPV L1 DNA were detected using PCR with MY09/MY11 primers in 31 specimens. All of them were recognized as BPV-1. Phylogenetic analysis has allowed the amplicons of partial L1 gene to be divided into 3 phylogenetic groups (A, B, C) and one separate isolate (20c). Sequence variants from phylogenetic groups B, C and isolate 20c represented new genetic variants of BPV-1 L1. Sequence variants from groups B and C were submitted to GenBank NCBI.

Distribution and chemical coding of calretinin- and calbindin-expressing enteric neurons in the duodenum of the sheep.

Recent decades has brought significant advances in our knowledge of the chemical coding and function of enteric neurons. Calcium ions are important second messenger involved in many aspects of neuron physiology. In the present study, we analyzed immunohistochemically the presence of calcium binding proteins (calretinin and calbindin) in various subpopulations of enteric neurons from the ovine duodenum. Ten percent of submucous neurons were immunoreactive (IR) to calretinin. The presence of calretinin was not detected in myenteric neurons. Calretinin-expressing nerve fibres were found in both myenteric and submucous ganglia, between the circular and longitudinal smooth muscle layers and in the lamina muscularis mucosae. Calretinin-IR submucous neurons did not exhibit the presence of SP, NPY and VIP. Co-localization of calretinin and serotonin was found only in a small number of submucous neurons. Calbindin was expressed in 35% of myenteric neurons and in 60% of submucous neurons. Nerve fibres containing calbindin were localized in myenteric and submucous ganglia where they frequently formed basket-like formations. Calbindin-positive nerve fibres emerging from myenteric ganglia ran between the circular and longitudinal smooth muscle layers. Immunoreactivity to calbindin was also visualized in the lamina muscularis mucosae, around mucosal glands and blood vessels. None of calbindin-IR myenteric neurons revealed immunoreactivity to SP, NPY, VIP and serotonin. Virtually all calbindin-expressing submucous neurons were SP-positive. In moderate numbers of submucous perikarya, co-incidence of calbindin and NPY, calbindin and VIP or calbindin and serotonin was observed. We conclude that in the ovine duodenum, the expression of calretinin and calbindin is species specific. Co-localization studies and distribution patterns indicate that in the duodenum of the sheep, calretinin and calbindin may be present in several functional subclasses of enteric neurons.

Distribution and chemical coding of intramural neurons in the porcine ileum during proliferative enteropathy.

Enteric neurons are highly adaptive in their response to various pathological processes including inflammation, so the aim of this study was to describe the chemical coding of neurons in the ileal intramural ganglia in porcine proliferative enteropathy (PPE). Accordingly, juvenile Large White Polish pigs with clinically diagnosed Lawsonia intracellularis infection (PPE; n=3) and a group of uninfected controls (C; n=3) were studied. Ileal tissue from each animal was processed for dual-labelling immunofluorescence using antiserum specific for protein gene product 9.5 (PGP 9.5) in combination with antiserum to one of: vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), somatostatin (SOM), neuropeptide Y (NPY) or galanin (GAL). In infected pigs, enteric neurons were found in ganglia located within three intramural plexuses: inner submucosal (ISP), outer submucosal (OSP) and myenteric (MP). Immunofluorescence labelling revealed increases in the number of neurons containing GAL, SOM, VIP and CGRP in pigs with PPE. Neuropeptides may therefore have an important role in the function of porcine enteric local nerve circuits under pathological conditions, when the nervous system is stressed, challenged or afflicted by disease such as PPE. However, further studies are required to determine the exact physiological relevance of the observed adaptive changes.

Re-examination of the topographical localization of facial nucleus in the pig.

Previous publications have provided different descriptions of the topographical organization of the facial nucleus of the pig. Since swine is used in biomedical research due to its embryological, anatomical and physiological similarities to human, we have reinvestigated the anatomical organization of the facial nucleus with application of fluorescent retrograde tracer Fast Blue, antibody to choline acetyltransferase and acetylcholinesterase histochemistry. Our findings demonstrate that in the porcine medulla facial motoneurons constitute a large cellular group occupying the ventro-lateral medulla. The neuronal group is interposed rostro-caudally between the superior and inferior olive, and located ventro-medially to the spinal nucleus of the trigeminal nerve. The present results clarify the anatomical description of this important brain stem nucleus in the pig.

Uterus-innervating neurones in porcine inferior mesenteric ganglion: an immunohistochemical characteristic.

The presence of tyrosine hydroxylase, neuropeptide Y, vasoactive intestinal polypeptide, galanin, Met-enkephalin-Arg-Gly-Leu, substance P and calcitonin gene-related peptide was studied with immunohistochemistry in uterus-innervating neurones found in the inferior mesenteric ganglia after fluorescent tracer (Fast Blue) injection into different regions of the porcine uterus (uterine cervix, paracervical, middle and paraoviductal part of the uterine horn). Virtually all Fast Blue-positive neurones found in the inferior mesenteric ganglia after tracer injection into all studied parts of the uterus contained tyrosine hydroxylase and ca. 45% of them contained neuropeptide Y. Single galanin-positive/Fast Blue-positive cells were found in the ganglia only after tracer injections into uterine cervix. No other studied substances were found in the Fast-Blue positive neurones of the inferior mesenteric ganglia.

Effect of total or partial uterus extirpation on uterus-projecting neurons in porcine inferior mesenteric ganglion. A. Changes in expression of transmitter-synthesizing enzymes--tyrosine hydroxylase, dopamine beta-hydroxylase and choline acetyltransferase.

Expression of tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH) and choline acetyltransferase (ChAT) was studied with immunohistochemistry, in situ hybridization, RT-PCR and immunoblotting in two populations of neurons of porcine inferior mesenteric ganglion (IMG) projecting to the uterine horn and uterine cervix after axotomy induced by partial or total uterus extirpation in sexually immature gilts. Uterus-projecting neurons of the IMG were identified by retrograde tracing with Fast Blue. Additionally, the distribution of ChAT-positive (ChAT+) and Met-enkephalin-positive (ME+) nerve fibers around uterus-projecting neurons was studied with immunohistochemistry. Immunohistochemistry detected that extirpation-induced axotomy reduced dramatically TH, but not DBH, expression in the uterus-projecting neurons, while the expression level of ChAT remained unchanged. Hybridization in situ performed with molecular probes for TH and ChAT confirmed these findings. RT-PCR did not detect any changes in the expression of TH and ChAT at mRNA level between control and hysterectomized animals. Immunoblotting did not detect significant changes in the expression of TH and DBH in IMG after partial or total extirpation. However, it detected that after total extirpation of the uterus a new form of ChAT with apparent lower molecular mass appears in the IMG of hysterectomized animals. It was found also that the number of ChAT+ and ME+ nerve fibers is lower around axotomy-affected neurons than around neurons in control gilts. The results presented here show clear axotomy-associated changes in the expression of TH, but not DBH and ChAT in the uterus-projecting neurons of the porcine IMG, as well as changes in the expression of ChAT and ME in the preganglionic nerve fibers.

Effect of total or partial uterus extirpation on sympathetic uterus-projecting neurons in porcine inferior mesenteric ganglion. B. Changes in expression of neuropeptide Y, galanin, vasoactive intestinal polypeptide, pituitary adenylate-cyclase activating peptide, somatostatin and substance P.

The expression of neuropeptide Y (NPY), galanin (GAL), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), somatostatin (SOM) and substance P (SP) was studied in the neurons of the inferior mesenteric ganglion (IMG) projecting to the uterine horn and uterine cervix after uterus extirpation-induced axotomy in sexually immature gilts. The expression was studied with immunohistochemistry, in situ hybridization and RT-PCR. Uterus-projecting neurons were identified by retrograde tracing with Fast Blue (FB). Immunohistochemistry revealed that FB-positive (FB+) uterus-projecting neurons in control animals contained only immunoreactivities to NPY (ca. 50%) and GAL (single neurons). Uterus extirpation increased the occurrence of NPY and GAL in FB+ neurons. No other studied neuropeptides were found in axotomized uterus-projecting neurons. Hybridization in situ revealed the reduction of NPY expression and induction of GAL expression in FB+ neurons. RT-PCR detected induction of GAL expression in the IMG after uterus extirpation. The expression level of NPY and SOM was significant and was not affected by axotomy. The expression level of PACAP was very low and did not differ between IMG of control, partially and totally hysterectomized animals. No VIP and SP expression was detected in all ganglia. The presented data show clear axotomy-related changes in the expression of GAL and NPY in the uterus-projecting neurons of the porcine IMG.

Effect of total or partial uterus extirpation on uterus-projecting neurons in porcine inferior mesenteric ganglion. C. Changes in expression of apoptosis-associated (Bcl-2 and Bax) and regeneration-associated (GAP-43) proteins.

The expression of Bcl-2 and Bax proteins was studied with immunohistochemistry, immuoblotting and RT-PCR in the uterine horn- and uterine cervix-projecting neurons of the inferior mesenteric ganglion (IMG) of the sexually immature gilts after partial or total hysterectomy. Additionally, the expression of regeneration-associated protein GAP-43 was studied in these neurons with immunohistochemistry. The uterus-projecting neurons were identified with retrograde fluorescent tracer Fast Blue (FB). The weak immunoreactivity to Bcl-2 and GAP-43 and moderately intense immunoreactivity to Bax was revealed in all FB+ (FB+) neurons of control and hysterectomized pigs. No difference in the intensity of immunostaining for Bcl-2, Bax and GAP-43 was found between control and hysterectomized gilts. Immunoblotting revealed the presence of Bcl-2 and Bax proteins in IMGs of control and hysterectomized animals and no difference in the band intensities between control and experimental groups was detected. RT-PCR detected weak induction of bcl-2 and bax only in the ganglia of animals which had undergone total hysterectomy. It was found that the axotomy of the uterus-projecting neurons located in the porcine IMG did not change the expression of the studied substances (Bcl-2, Bax and GAP-43) at protein level and only the induction of bcl-2 and bax at the level of RNA was visible.

Adrenergic and cholinergic innervation of the mammary gland in the pig.

Adrenergic and acetylcholinesterase-positive (AChE-positive) innervation of the mammary gland in the sexually immature and mature pigs was studied using histochemical methods. Upon examining the adrenergic and cholinergic innervation, the adrenergic innervation was found to be much more developed. The majority of both sub-populations of the nerve fibres studied was localized in the subcutaneous tissue of the mammary gland. Adrenergic and AChE-positive nerve fibres also supplied structures of the nipple (subcutaneous tissue, blood vessels, smooth muscles fibres) and glandular tissue (blood vessels, lactiferous ducts). The glandular tissue contained the smallest number of adrenergic and AChE-positive nerve fibres. No distinct differences were observed in the adrenergic and AChE-positive innervation of the porcine mammary gland between the juvenile and non-pregnant adult animals.

Immunohistochemical study of the otic ganglion in the pig.

The presence of choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), substance P (SP) and calcitonin gene-related peptide (CGRP) was studied in neurons and nerve fibers of the porcine otic ganglion. ChAT-positive neurons were very numerous while VAChT-positive nerve cells were moderate in number. The number of neurons containing NPY and VIP was lower and those containing SOM, GAL, SP or CGRP were observed as scarce, or single nerve cells. The above mentioned substances (except SOM) were present in nerve fibers of the ganglion. ChAT- and VAChT-positive nerve fibers were numerous, while the number of nerve terminals containing NPY, VIP and SP was lower. GAL- and CGRP-positive nerve fibers were scarce.

Influence of active immunization against GnRH on VIP- and NPY-positive innervation of the porcine testis.

The influence of an anti-GnRH vaccine on VIP- and NPY-positive innervation of testes was studied in the pig. The immunization prevented the occurrence of changes in the pattern of VIP- and NPY-positive testicular innervation associated with the sexual maturation: it maintained the density of innervation at the high level characteristic for sexually immature animals. The effect was dependent on the method of immunization: the application of two doses of the vaccine was more efficient than application of only one dose, and vaccination with adjuvant was more efficient than vaccination with the plain vaccine. The studies on VIP and NPY concentration in the testicular tissue with radioimmunoassay (RIA) revealed immunization-dependent changes in the peptide concentration, however, some discrepancies between morphological changes and peptide levels were observed.