Intrinsic Innervation of the Persian Squirrel (Sciurus anomalus) Ileum

Most investigations related to the characterisation of the enteric nervous system (ENS) are pivoted on the intestine of small rodents, but few studies are available on the ENS of wild or ‘unconventional’ rodents. Anti‐PGP 9.5 and anti‐Hu antibodies were utilised to recognise the distribution pattern of neuronal cell bodies and fibres of the ileum of the Persian squirrel (Sciurus anomalus) ENS. The percentages of subclasses of enteric neurones in the total neuronal population were investigated by neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene‐related peptide (CGRP), substance P (SP), and calbindin (CALB). Myenteric plexus (MP) and submucosal plexus (SMP) neurones showing nNOS immunoreactivity (IR) were 41 ± 4% and 11 ± 6%, respectively, whereas cells expressing ChAT‐IR were 56 ± 9% and 74 ± 16%, respectively. nNOS‐IR was co‐expressed by 21 ± 2% and 9 ± 4% of the MP and SMP cholinergic neurones, respectively, whereas the nNOS‐IR MP and SMP neurones co‐expressing ChAT‐IR were 86 ± 6% and 89 ± 2%, respectively. CGRP‐IR and SP‐IR were expressed, respectively, by 13 ± 5% and 6 ± 3% of MP and 18 ± 2% and 2 ± 2% of SMP neurones. CALB‐IR was expressed by 22 ± 8% and 56 ± 14% of MP and SMP neurones, respectively. MP and SMP cholinergic neurones co‐expressed nNOS‐IR (21 ± 2% and 9 ± 4%, respectively) and a very high percentage of nNOS‐IR neurones showed ChAT‐IR (86 ± 6% and 89 ± 2%, respectively). MP and SMP CALB‐IR neurones co‐expressed ChAT‐IR (100% and 63 ± 11%, respectively) and CGRP‐IR (89 ± 5% and 26 ± 7%, respectively). Our data might contribute to the neuroanatomical knowledge of the gastrointestinal tract in exotic mammals and provide a comparison with the available data on other mammals.     

Intrinsic innervation of the horse ileum

This paper describes the morphology and distribution of the enteric nervous system (ENS) cells and fibres immunoreactive for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene-related peptide (CGRP), NF200 kDa (NF200), and S100 protein. The percentages of subclasses of enteric neurons in the total neuronal population were investigated by the use of anti-PGP 9.5 or anti-NSE antibodies.ChAT-IR myenteric plexus (MP) and submucosal plexus (SMP) neurons were 66 ± 7% and 74 ± 15%, respectively, whereas those cells expressing nNOS-IR were 38 ± 7% and 5 ± 1%, respectively. MP and SMP neurons expressing both phenotypes were also present. SP-IR was expressed by 14 ± 13% of MP and 66 ± 8% of SMP neurons whereas CGRP-IR was observed only in the SMP (43 ± 6%). NF200-IR was expressed by 61 ± 15% and 91 ± 10% of the MP and SMP neurons, respectively. The majority of the CGRP-IR SMP neurons expressed also SP-IR. Almost all SP-IR neurons in both the plexuses were cholinergic. The present study quantifies the main neuronal subpopulations of the ENS of the horse ileum; these data might be utilized to understand the neuronal modifications which occur in several gastrointestinal tract disorders.     

Chemical Coding of Sensory Neurons Supplying the Hip Joint Capsule in the Sheep

Immunohistochemical properties of nerve fibres supplying the joint capsule were previously described in many mammalian species, but the localization of sensory neurons supplying this structure was studied only in laboratory animals, the rat and rabbit. However, there is no comprehensive data on the chemical coding of sensory neurons projecting to the hip joint capsule (HJC). The aim of this study was to establish immunohistochemical properties of sensory neurons supplying HJC in the sheep. The study was carried out on 10 sheep, weighing about 30–40 kg. The animals were injected with a retrograde neural tracer Fast Blue (FB) into HJC. Sections of the spinal ganglia (SpG) with FB‐positive (FB+) neurons were stained using antibodies against calcitonin gene‐related peptide (CGRP) substance P (SP), pituitary adenylate cyclase‐activating peptide (PACAP), nitric oxide synthase (n‐NOS), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), Leu‐5‐enkephalin (Leu‐Enk), galanin (GAL) and vesicular acetylcholine transporter (VACHT). The vast majority of FB+ neurons supplying HJC was found in the ganglia from the 5th lumbar to the 2nd sacral. Immunohistochemistry revealed that most of these neurons were immunoreactive to CGRP or SP (80.7 ± 8.0% or 56.4 ± 4.8%, respectively) and many of them stained for PACAP or GAL (52.9 ± 2.9% or 50.6 ± 19.7%, respectively). Other populations of FB+ neurons were those immunoreactive to n‐NOS (37.8 ± 9.7%), NPY (34.6 ± 6.7%), VIP (28.7 ± 4.8%), Leu‐Enk (27.1 ± 14.6) and VACHT (16.7 ± 9.6).     

Sources of Sensory Innervation of the Hip Joint Capsule in the Rabbit – A Retrograde Tracing Study

The aim of the study was to investigate the sensory innervation of the hip joint capsule in the rabbit. Individual animals were injected with retrograde fluorescent tracer Fast Blue (FB) into the lateral aspect of the left hip joint capsule (group LAT, n = 5) or into the medial aspect of the hip joint capsule (group MED, n = 5), respectively. FB‐positive (FB+) neurons were found within ipsilateral lumbar (L) and sacral (S) dorsal root ganglia (DRG) from L7 to S2 (group LAT) and from L6 to S4 (group MED). They were round or oval in shape with a diameter of 20–90 μm. The neurons were evenly distributed throughout the ganglia. The average number of FB+ neurons was 16 ± 2.8 and 27.6 ± 3.5 in rabbits from LAT and MED, respectively. The largest average number of FB+ neurons in animals of group LAT was found within the S1 DRG (8 ± 1.7), while S2 ganglion contained the smallest number of the neurons (3.6 ± 1). In the L7 DRG, the average number of FB+ neurons was 6.2 ± 1.6. In rabbits of MED group, the largest number of FB+ neurons was found within the S1 DRG (13.4 ± 4), while the smallest one was found within the S3 ganglion (1.4 ± 0.4). In L6, L7, S2 and S4 ganglia, the number of retrogradely labelled neurons amounted to 1.6 ± 0.5, 4 ± 1.5, 4.4 ± 1.5 and 2.8 ± 1.7, respectively. The data obtained can be very useful for further investigations regarding the efficacy of denervation in the therapy of hip joint disorders in rabbits.     

Expression of β2 adrenoceptors within enteric neurons of the horse ileum

The activity of the gastrointestinal tract is regulated through the activation of adrenergic receptors (ARs). Since data concerning the distribution of ARs in the horse intestine is virtually absent, we investigated the distribution of β₂-AR in the horse ileum using double-immunofluorescence. The β₂-AR-immunoreactivity (IR) was observed in most (95%) neurons located in submucosal plexus (SMP) and in few (8%) neurons of the myenteric plexus (MP). Tyrosine hydroxylase (TH)-IR fibers were observed close to neurons expressing β2-AR-IR. Since β₂-AR is virtually expressed in most neurons located in the horse SMP and in a lower percentage of neurons in the MP, it is reasonable to retain that this adrenergic receptor could regulate the activity of both secretomotor neurons and motor neurons innervating muscle layers and blood vessels. The high density of TH-IR fibers near β₂-AR-IR enteric neurons indicates that the excitability of these cells could be directly modulated by the sympathetic system.     

Extrinsic afferents supplying the ovine duodenum and ileum

The study aimed at establishing the distribution of primary sensory neurons by means of retrograde tracers Diamidino Yellow (DY) and Fast Blue (FB) injected into both the sheep duodenum and ileum, respectively. Many DY-labelled cells were found in both the distal vagal ganglia (DVG) and the spinal ganglia (SG) from T9–L3; on the contrary, the majority of the FB-labelled cells were found in the SG. In the SG, a double immunofluorescence stain was used to reveal Nitric Oxide Synthase-Immunoreactivity (NOS-IR) in association with: substance P (SP), calcitonin gene-related peptide (CGRP), neurofilament 200 kDa (NF) and isolectin B₄ (IB4). The labelled neurons, both DY and FB generally ranged in size from medium to large. The majority of the SG duodenal projections were NOS negative; the majority of the SG ileal afferent neurons expressed NOS-IR. Both DY and FB NOS-IR neurons often co-localized IB4, CGRP and SP, but rarely NF.     

The Distribution and Chemical Coding of Intramural Nerve Structures Supplying the Porcine Stomach

The present study investigated the arrangement and chemical coding of intramural nerve structures supplying the porcine stomach. Tissue samples comprising all layers of the wall of the ventricular fundus were collected from juvenile female pigs (n = 4), which were first deeply anaesthetized and then transcardially perfused with buffered paraformaldehyde. The cryostat sections were processed for double‐labelling immunofluorescence to study the distribution of the intramural nerve structures (visualized with antibodies against protein gene‐product 9.5) and their neurochemical characteristics using antibodies against vesicular acetylcholine transporter (VAChT), nitric oxide synthase (NOS), galanin (GAL), vasoactive intestinal‐polypeptide (VIP), somatostatin (SOM) and substance P (SP). The study confirmed the presence of three distinct nerve plexuses within the wall of the porcine stomach including one myenteric plexus and two, outer and inner, submucous plexuses. The outer and inner submucous plexuses (OSP and ISP, respectively) were similar in respect to the chemical coding of neurons they contained. Most of the neurons expressed immunoreactivity to SP (ISP 58%; OSP 60%) or to VAChT (ISP 56%; OSP 56%), some of them stained for GAL (ISP 18%; OSP 15%) and solitary nerve cells were SOM‐positive (in ISP only). No neurons in the submucous plexuses displayed immunoreactivity to VIP or NOS. In the myenteric plexus, some neurons stained for NOS (20%), VAChT (15%), GAL (10%), VIP (8%) or SP (8%) while no neurons immunoreactive for SOM were encountered. In both submucous and myenteric plexuses, many varicose nerve fibres expressed immunoreactivity to VAChT, GAL or SP, while VIP‐, SOM‐ or NOS‐positive nerve terminals were less numerous. The comparison of the present results with those obtained by other authors has revealed distinct inter‐species differences regarding the arrangement and chemical coding of nerve structures supplying the mammalian stomach.     

GAL‐ and VIP‐Immunoreactive Nerve Structures in the Ileum and Large Intestine of Pigs with Dysentery

The morphology, neurochemistry and function of intramural nerve structures in the mammalian gastrointestinal tract are relatively well known, but in normal, healthy individuals. The present study was aimed at investigating the chemical coding of nerve structures in the wall of the ileum and large intestine in normal pigs (n = 3) and in pigs undergoing dysentery (n = 6). Dysentery was evoked by artificial infection of the clinically healthy animals per os with Brachyspira hyodysenteriae. All the animals were deeply anaesthetized and transcardially perfused with 4% paraformaldehyde. The cryostat sections of the intestines were processed for double‐labelling immunohistochemistry using antisera against PGP 9.5, GAL and VIP. In the intramural plexuses of the control pigs, the percentage of GAL‐immunoreactive (GAL‐IR) perykarya varied from 11% (descending colon) to 19% (centrifugal turns of the ascending colon) whereas in the dysenteric pigs, it was distinctly higher, reaching from 28% (ileum) up to 48% (cecum). In the control animals, the percentage of VIP‐IR neuronal somata varied from 3% (descending colon) to 19% (ileum). In dysenteric pigs, it was from 6% (descending colon) up to 28% (cecum). In the muscular coat (MC) and mucous membrane (MM) of the normal intestine, very numerous GAL‐ and VIP‐IR nerve fibres were observed. The nerve fibres in the myenteric plexus (MP) were even more numerous than those in the muscular coat while in the outer (OSP) and inner (ISP) submucous plexuses, they were less abundant. In the dysenteric pigs, the nerve fibres found in MC, MP and OSP were less numerous, whereas those observed in ISP and MM were more abundant than those in the control animals. The present results suggest that GAL and VIP are involved in the regulation of inflammatory processes developing in the porcine gastrointestinal tract during dysentery.     

Immunohistochemical properties of motoneurons supplying the trapezius muscle in the rat

Combined retrograde tracing (using fluorescent tracer Fast blue) and double-labelling immunofluorescence were used to study the distribution and immunohistochemical characteristics of neurons projecting to the trapezius muscle in mature male rats (n = 9). As revealed by retrograde tracing, Fast blue-positive (FB+) neurons were located within the ambiguous nucleus and accessory nucleus of the grey matter of the spinal cord. Immunohistochemistry revealed that nearly all the neurons were cholinergic in nature [choline acetyltransferase (ChAT)-positive]. Retrogradely labelled neurons displayed also immunoreactivities to calcitonin gene-related peptide (CGRP; approximately 60% of FB+ neurons), nitric oxide synthase (NOS; 50%), substance P (SP; 35%), Leu5-Enkephalin (LEnk; 10%) and vasoactive intestinal polypeptide (VIP; 5%). The analysis of double-stained tissue sections revealed that all CGRP-, VIP- and LEnk-immunoreactive FB+ perikarya were simultaneously ChAT-positive. The vast majority of the neurons expressing SP- or NOS-immunoreactivity were also cholinergic in nature; however, solitary somata were ChAT-negative. FB+ perikarya were surrounded by numerous varicose nerve fibres (often forming basket-like structures) immunoreactive to LEnk or SP. They were also associated with some CGRP-, NOS- and neuropeptide Y-positive nerve terminals.     

Influences of protein ingestion on glucagon‐like peptide (GLP)‐1‐immunoreactive endocrine cells in the chicken ileum

Influences of a specific dietary nutrient on glucagon‐like peptide (GLP)‐1‐containing cells in the chicken intestine are not yet clear. Significance of dietary protein level on GLP‐1‐containing cells in the chicken ileum was investigated. Chickens fed control or experimental diets of varying protein levels were examined using immunohistochemical and morphometrical techniques. We show that the protein ingestion had an impact on the activities of GLP‐1‐immunoreactive cells in the chicken ileum. Weight gains declined with decreasing dietary crude protein (CP) levels, but no significant differences were detected in the daily feed intake and villous height. GLP‐1‐immunoreactive cells with a round or oval shape were frequently observed in the lower CP level groups (4.5% and 0%). Frequencies of occurrence of GLP‐1‐immunoreactive cells were 41.1 ± 4.1, 38.5 ± 4, 34.8 ± 3.1 and 34.3 ± 3.7 (cells/mm², mean ± SD) for dietary CP level of 18%, 9%, 4.5% and 0% groups, respectively and significant differences were recognized between the control and lower CP level groups (P < 0.05). Multiple regression analysis indicated a significant correlation between the daily protein intake and frequencies of occurrence of GLP‐1‐immunoreactive cells. The protein ingestion is one of the signals that influence GLP‐1‐containing cells in the chicken small intestine.     

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.     

Effect in sheep of dietary concentrate content on secretion of growth hormone, insulin and insulin-like growth factor-I after feeding

This study was designed to examine the effects of the proportion of concentrate in the diet on the secretion of growth hormone (GH), insulin and insulin‐like growth factor‐I (IGF‐I) secretion and the GH‐releasing hormone (GHRH)‐induced GH response in adult sheep fed once daily. Dietary treatments were roughage and concentrate at ratios of 100:0 (0% concentrate diet), 60:40 (40% concentrate diet), and 20:80 (80% concentrate diet) on a dry matter basis. Mean plasma concentrations of GH before daily feeding (10.00–14.00 hours) were 11.4 ± 0.4, 10.1 ± 0.5 and 7.5 ± 0.3 ng/mL on the 0, 40 and 80% concentrate diet treatments, respectively. A significant decrease in plasma GH concentration was observed after daily feeding of any of the dietary treatments and these decreased levels were maintained for 8 h (0%), 12 h (40%) and 12 h (80%), respectively (P < 0.05). Plasma IGF‐I concentrations were significantly decreased 8–12 h and 4–16 h after the end of feeding compared with the prefeeding level in the 40 and 80% concentrate diet treatments, respectively (P < 0.05). GHRH injection brought an abrupt increase in the plasma GH concentrations, reaching a peak 10 min after each injection, but, after the meal, the peak plasma GH values for animals fed 40% (P < 0.05) and 80% (P < 0.01) concentrate diet were lower than that for roughage fed animals. The concentrate content of a diet affects the anterior pituitary function of sheep resulting in reduced baseline concentrations of GH and prolonged GH reduction after feeding once daily.     

Preparation,characterization, and pharmacokinetics of tilmicosin‐ and florfenicol‐loaded hydrogenated castor oil‐solid lipid nanoparticles

To effectively control bovine mastitis, tilmicosin (TIL)‐ and florfenicol (FF)‐loaded solid lipid nanoparticles (SLN) with hydrogenated castor oil (HCO) were prepared by a hot homogenization and ultrasonication method. In vitro antibacterial activity, properties, and pharmacokinetics of the TIL‐FF‐SLN were studied. The results demonstrated that TIL and FF had a synergistic or additive antibacterial activity against Streptococcus dysgalactiae, Streptococcus uberis, and Streptococcus agalactiae. The size, polydispersity index, and zeta potential of nanoparticles were 289.1 ± 13.7 nm, 0.31 ± 0.05, and ?26.7 ± 1.3 mV, respectively. The encapsulation efficiencies for TIL and FF were 62.3 ± 5.9% and 85.1 ± 5.2%, and the loading capacities for TIL and FF were 8.2 ± 0.6% and 3.3 ± 0.2%, respectively. The TIL‐FF‐SLN showed no irritation in the injection site and sustained release in vitro. After medication, TIL and FF could maintain about 0.1 μg/mL for 122 and 6 h. Compared to the control solution, the SLN increased the area under the concentration–time curve (AUC_0‐t), elimination half‐life (T_½ke), and mean residence time (MRT) of TIL by 33.09‐, 23.29‐, and 37.53‐fold, and 1.69‐, 5.00‐, and 3.83‐fold for FF, respectively. These results of this exploratory study suggest that the HCO‐SLN could be a useful system for the delivery of TIL and FF for bovine mastitis therapy.     

Proinflammatory Cytokine and Chemokine Gene Expression Profiles in Subcutaneous and Visceral Adipose Tissue Depots of Insulin‐Resistant and Insulin‐Sensitive Light Breed Horses

Background: Insulin resistance has been associated with risk of laminitis in horses. Genes coding for proinflammatory cytokines and chemokines are expressed more in visceral adipose tissue than in subcutaneous adipose tissue of insulin‐resistant (IR) humans and rodents. Hypothesis/Objectives: To investigate adipose depot‐specific cytokine and chemokine gene expression in horses and its relationship to insulin sensitivity (SI). Animals: Eleven light breed mares. Methods: Animals were classified as IR (SI = 0.58 ± 0.31 × 10^?4 L/min/mU; n = 5) or insulin sensitive (IS; SI = 2.59 ± 1.21 × 10^?4 L/min/mU; n = 6) based on results of a frequently sampled intravenous glucose tolerance test. Omental, retroperitoneal, and mesocolonic fat was collected by ventral midline celiotomy; incisional nuchal ligament and tail head adipose tissue biopsy specimens were collected concurrently. The expression of tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐1β, IL‐6, plasminogen activator inhibitor‐1 (PAI‐1), and monocyte chemoattractant protein‐1 (MCP‐1) in each depot was measured by real‐time quantitative polymerase chain reaction. Data were analyzed by 2‐way analysis of variance for repeated measures (P < .05). Results: No differences in TNF‐α, IL‐1β, IL‐6, PAI‐1, or MCP‐1 mRNA concentrations were noted between IR and IS groups for each depot. Concentrations of mRNA coding for IL‐1β (P= .0005) and IL‐6 (P= .004) were significantly higher in nuchal ligament adipose tissue than in other depots. Conclusions and Clinical Importance: These data suggest that the nuchal ligament depot has unique biological behavior in the horse and is more likely to adopt an inflammatory phenotype than other depots examined. Visceral fat may not contribute to the pathogenesis of obesity‐related disorders in the horse as in other species.     

The equine enteric nervous system — Neuron characterization and distribution in adults and juveniles

A study of myenteric and submucosal plexuses was undertaken in the jejunum and ileum of horses and ponies in which no clinical or pathological evidence of intestinal abnormality was apparent. Complete transverse sections of the intestine, stained by a modified haematoxylin and eosin method, were examined using up to 20 sequential sections per animal. Information was gathered from adult, juvenile and fetal equidae. In adults, the longitudinal muscle layers were thinner than the circular muscle layers and the ileum had thicker layers compared to the jejunum. In adults, the submucosal plexus had more neurons per section than the myenteric plexus by mean ratios of 1:3 in the jejunum and 1:1.9 in the ileum. In juveniles, the ratios were respectively 1:1.8 and 1:1.5 and in the fetus 1:2.5 and 1:1.3. The three-dimensional distribution of neurons in both plexuses varied from animal to animal and no consistent pattern was observed. Groups of neurons contained between one and 42 cells per section examined and their length in a cranio-caudal direction varied from 10 to over, 100 µm. There were few statistical differences observed between the cranial, middle and caudal portions of either the jejunum or the ileum when neuron groups or neuron numbers per section were examined in 10 adult animals.Abbreviations H&E haematoxylin and eosin Deceased; formerly of the Moredun Research Institute, 408, Gilmerton Road, Edinburgh, EH17 7JH, UK     

Immunohistochemical Characterization of Neurones in the Hypoglossal Nucleus of the Pig

With 4 figures and 1 table In this study, the presence of several neurotransmitters and transmitter synthesizing enzymes was studied in hypoglossal nucleus (HN) of the juvenile (4 months old) female pigs (n = 3). Double‐labeling immunofluorescence revealed neurones expressing cholinacetyltranspherase (ChAT), calcitonin gene‐related peptide (CGRP), nitric oxide synthase (NOS), and somatostatin (SOM). Nerve fibers within HN were ChAT, CGRP, NOS, SOM, substance P (SP), Leu‐5‐enkephalin (Leu‐5‐Enk), ß‐dopamine hydroxylase (DßH), neuropeptide Y (NPY) positive. Virtually all the perikarya contained ChAT, whereas CGRP was present in 47% of the neurones. Nerve cell bodies containing NOS or SOM were only occasionally observed. Immunoreactive nerve fibers were found in a close vicinity of the perikarya, often forming baskets around nerve cell bodies. The results obtained were compared with similar data obtained in other species. The presence of immunoreactive structures, origin of the nerve fibers, and functional significance of the findings are discussed.     

Influence of Inseminate Components on Porcine Leucocyte Migration In Vitro and In Vivo after Pre- and Post-Ovulatory Insemination

A post‐breeding migration of leucocytes (PMN) into the uterus is considered to be an important reason for sperm losses. Minimizing such effects may be necessary for successful insemination with low sperm numbers, as required with sex‐sorted spermatozoa. We examined the magnitude of PMN influx 3 h after pre‐ or post‐ovulatory insemination with various combinations of seminal plasma (SP), semen extender Androhep? (AH; Minitüb, Tiefenbach, Germany) and sperm preparations (S). Pre‐ovulatory inseminations with preparations containing 98% AH caused a massive influx of PMN, independent of whether spermatozoa were present (628 ± 189 × 10⁶ leucocytes/uterine horn) or not (580 ± 153 × 10⁶). Post‐ovulatory, 98% AH caused a comparable immigration only in the absence of sperm cells (AH: 569 ± 198 × 10⁶, AH+S: 162 ± 102 × 10⁶). The presence of SP significantly dampened the numbers of recruited uterine leucocytes. The reaction to all inseminates containing 98% SP both with and without spermatozoa, used before ovulation (SP: 14 ± 6 × 10⁶, SP+S: 73 ± 27 × 10⁶) and after ovulation (SP: 60 ± 32 × 10⁶, SP+S: 51 ± 33 × 10⁶) did not differ significantly from controls using phosphate buffered saline (PBS) (pre‐ovulatory: 1 ± 1 × 10⁶, post‐ovulatory: 11 ± 9 × 10⁶). Quantitative in vitro transmigration assays with blood‐derived PMN proved that AH‐induced leucocyte migration into the uterus to be not as a result of direct chemotaxis, because, on account of the chelator citrate, AH significantly inhibited the transmigration towards recombinant human Interleukin‐8 (rhCXCL8) (AH: 14 ± 5% migration rate vs controls: 37 ± 6%, p < 0.05). Supernatants of spermatozoa incubated in PBS for 1, 12 or 24 h showed neither chemoattractive nor chemotaxis‐inhibiting properties. SP at ≥0.1% [v/v] significantly inhibited the in vitro transmigration of PMN. With respect to in vivo migration of neutrophils, the striking difference in the results between semen extender and seminal plasma suggests that adaptation of extender composition is needed to reflect more closely the in vivo regulatory potential of natural seminal plasma.