Reproductive immunology in viviparous mammals: evolutionary paradox of interactions among immune mechanisms and autologous or allogeneic gametes and semiallogeneic foetuses.

Literally, reproductive immunology was born in bovine on-farm reproduction where seminal experiments intended for developing methods for embryo transfer in cattle were performed. Actually, these experiments led to two of major concepts and fundamental principles of reproductive immunology using the bovine species as a model for biomedical research, namely the concept of acquired immunological tolerance and the paradox of the semiallogeneic bovine foetus whereby such organism can develop within an immunologically competent host. Peter Medawar, a scientist who together with Frank Macfarlande Burnet shared the 1960 Nobel Prize in physiology or medicine for discovery of acquired immunological tolerance, while studying dizygotic cattle twins, thereby giving birth to reproductive immunology. Also, these findings significantly influenced development of organ transplants and showed that using farm animals as models for studying transplantation immunology had general relevance for mammalian biology and health including those of humans. However, the interest for further research of the fascinating maternal immune influences on pregnancy and perinatal outcomes and of the prevention and treatment of immunologically mediated reproductive disorders in viviparous mammals of veterinary relevance by veterinary immunologists and reproductive clinicians have been very scarce regarding the application of nonspecific immunomodulatory agents for prevention and treatment of subfertility and infertility in pigs and cattle, but still broadening knowledge in this area and hold great potential for improving such therapy in the future. The aim of the current overview is to provide up-to-date information and explaining/translating relevant immunology phenomena into veterinary practice for specialists and scientists/clinicians in reproduction of animals.

Effects of dietary clinoptilolite on reproductive performance, serum progesterone and insulin-like growth factor-1 concentrations in dairy cows during pregnancy and lactation.

The objective of this study was to evaluate the effects of dietary zeolite clinoptilolite on re- productive performance, serum progesterone and insulin-like growth factor 1 (IGF-1) concentra- tions in 78 Holstein Friesian (HF) cows during pregnancy and lactation. The cows were divided into two groups comprising 40 (control group; CON) and 38 (CPL group) cows. To assess repro- ductive performance of HF cows the following variables were registered: the interval from calv- ing to first insemination (days open to first service, DFS), the interval from calving to pregnancy (days open to pregnancy, DOP), and the number of services per pregnancy (NSP). The average values of progesterone (5.64±0.59 ng/mL vs. 5.16±0.64 ng/mL) were not statistically different (p⟨0.05) and IGF-1 levels (400.17±17.72 ng/mL vs. 348.36±20.39 ng/mL) were higher in the CON than in the CPL group which received 50 g of clinoptilolite twice a day. However, in the CPL group ovarian cyclity resumed on days 40 and 60 postpartum. In addition, DFS (p⟨0.05) and DOP (p>0.05) were shorter in the CPL than in the CON group (115.1±19.9 and 137.5±36.3 days vs. 124.2±17.3 and 143.8±33.5 days, respectively). During 305 days of lactation, milk production was higher in the CPL vs. CON (8325.5±628.8 kg vs. 8050±586.8 kg). The NSP was lower in the CPL than in CON group (1.91 vs. 2.14). The dietary clinoptilolite supplement had a positive in- fluence on milk yield, exhibited modulating effects on endocrine status of dairy cows, and im- proved reproductive performance, with the decreased NSP, and fewer DFS and DOP.

The influence of exogenous melatonin on antioxidative status in seminal plasma and spermatozoa in French Alpine bucks during the nonbreeding season.

Research data regarding exogenous melatonin effects on buck semen antioxidative status during the breeding and nonbreeding seasons are scarce or missing. The aim of this study was to investigate the effect of melatonin implants on the activity and ratios of antioxidative enzymes and on malondialdehyde concentrations in the seminal plasma and spermatozoa of French Alpine bucks during the 3 mo of the nonbreeding season. Two groups of randomly assigned bucks were either treated with melatonin (n = 6) or served as controls (n = 6). Semen samples were collected weekly, seminal plasma was separated from spermatozoa, and activities of glutathione peroxidase (GSH-Px), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), and the concentration of malondialdehyde were determined. The melatonin-treated bucks had significantly higher CAT/SOD and GSH-Px/SOD ratios, but a significantly lower GR/GSH-Px ratio in seminal plasma and spermatozoa during the different periods of the nonbreeding season. In conclusion, exogenous melatonin influenced the activities of all ratios and some single antioxidative enzymes tested in bucks' seminal plasma and spermatozoa. Here we provide the first evidence derived from an intervention, that is, melatonin treatment, which affects antioxidative enzymes ratios in the semen of bucks, and could thus be recommended as an indicator of oxidative stress severity and antioxidative status of semen.

Dietary supplementation with mannan oligosaccharide and clinoptilolite modulates innate and adaptive immune parameters of weaned pigs.

The aim of this study was to assess modulatory effects of dietary supplements mannan oligosaccharide (MOS) and clinoptilolite (CPL) as potential alternatives to antibiotic growth promoters (AGP) given to 4-week old pigs at weaning (Day 0) on their innate/adaptive immunity by determining: alterations in C-reactive protein (CRP) and haptoglobin (HpG) serum levels, efficiency of blood monocytes (MO) and neutrophilic granulocytes (GR) for in vitro phagocytosis (PHC)/microbicidity (MBC) and proportion of extrathymic double positive CD4 CD8 (CD4+CD8+) T cells throughout 35 days of the study. Neither MOS nor CPL changed the serum concentrations of CRP, whereas that of HpG was significantly increased in the CPL supplemented pigs (p<0.05) at Day 35. Activity of PHA of GR was significantly increased by both dietary supplements (p<0.05) from Day 7 to Day 35. Also, the GR from pigs fed with both supplements had significantly increased MBC at Day 7 (p<0.05), but at Day 35 such an increase was observed only for CPL. The in vitro PHC/MBC of MO did not change in either group of supplemented pigs. The pigs supplemented with MOS had a significantly higher proportion of CD4+CD8+ T lymphocytes at Day 28 (p<0.05). Although both supplements showed a promising ability to stimulate rather innate than adaptive cellular immunity, it does not appear that any solely applied natural substance such as MOS or CPL in the current study could be a competitive alternative to conventional AGP for improving health and promoting growth in weaned pigs.

Age-related differences of semen quality, seminal plasma, and spermatozoa antioxidative and oxidative stress variables in bulls during cold and warm periods of the year.

The aims of this study were to determine the presence and quantities of antioxidative status and oxidative stress (OS) variables in the seminal plasma and spermatozoa of bulls of varying age during cold and warm periods of the year, and to establish the correlation of these variables with semen quality parameters. The study was conducted on two groups each comprising nine Simmental bulls: one group contained younger animals (aged 2 to 4 years) and the second older animals (aged 5 to 10 years). Semen samples were collected using an artificial vagina for biochemical analysis. Seminal plasma and spermatozoa activities of total superoxide dismutase (TSOD), manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), catalase (CAT), selenium-dependent glutathione peroxidase, reduced glutathione and concentrations of total protein (TP), thiobarbituric acid reactive substances (TBARS) and protein carbonyl content (PCC) were determined. Several antioxidants in seminal plasma were also determined: total glutathione peroxidase (TGSH-Px), selenium-independent glutathione peroxidase (Non-SeGSH-Px), uric acid, albumins (ALB) and alkaline phosphatase (ALP). Significantly higher spermatozoa motility was observed during the cold v. warm period, and a significantly higher volume and total number of spermatozoa per ejaculate was observed in older than in younger bulls. Significantly higher values of ALP, TP and ALB were found in seminal plasma of older bulls than in younger bulls during the warm period. The seminal plasma of younger bulls showed significantly higher activities of TSOD, MnSOD, CuZnSOD, TGSH-Px and Non-SeGSH-Px. Younger bulls had significantly higher PCC concentration and activity of CAT in seminal plasma than older bulls during the cold period. Significantly higher concentrations of PCC and TBARS, and activities of TSOD, MnSOD and CuZnSOD were established in spermatozoa of the younger than in older bulls during the warm period. It could be concluded that antioxidative and OS variables differ significantly depending on bull age and time of year. Younger bulls were more sensitive to elevated ambient temperatures during the warm period, when the higher enzymatic antioxidative protection in seminal plasma and spermatozoa were insufficient to counteract the intensive oxidative processes in spermatozoa, which eventually resulted in decreased spermatozoa motility. The estimation of antioxidative and OS variables in seminal plasma and spermatozoa may have practical value for the assessment of bull semen quality.

Monitoring of libido and semen quality parameters in melatonin-treated French alpine bucks during the non-breeding season.

The aim of this study was to investigate the effects of exogenous melatonin on libido and semen quality parameters in bucks during the non-breeding season. Twelve bucks of the French alpine breed from 1.5 to 4 years of age were assigned into melatonin (MG) and control (CG) groups, with 6 bucks in each group. The experimental period was 3 months (March-May), divided into six periods of 15 days each. The bucks in the MG group received four melatonin implants at the end of March. Two semen samples were taken from the bucks by artificial vagina once per week and their libido estimated. Volume and spermatozoa concentration, their mass motility and motility, proportion of live and total abnormal and forms with abnormal head and tail were determined in the obtained ejaculate samples. The total number of spermatozoa and functional spermatozoa fraction in the ejaculate was also calculated. The MG bucks had significantly higher mass motility and motility of spermatozoa in the first half of April, and a higher proportion of live spermatozoa in the first and second half of April (p < .05). Differences in libido intensity were not significant. The results indicated that the application of melatonin significantly improved the qualitative parameters of semen in bucks, as seen in increased mass motility, motility of spermatozoa and proportion of live spermatozoa shortly following melatonin insertion. Therefore, the results of the current study are novel regarding the use of melatonin treatment during the non-breeding season to improve the qualitative parameters of ejaculates in bucks.

The onset of puberty in Cameroon Dwarf goats kept as pets in northwestern Croatia.

The aim of this study was to determine onset of puberty in Cameroon Dwarf goats (CDGs) kept as pets in northwestern Croatia by determining progesterone (PGS) and insulin-like growth factor-I (IGF-I) levels in the blood by RIA methods. The first cyclic ovarian activity was estimated according to hormone profiles as determined in CDG in a moderate climate environment. Sixteen female CDG kids were kept in stables with access to pasture which provided space for exercise. The goat kids born in winter (December-January-February), spring (March-April-May), summer (June-July-August) and autumn (September-October-November) were assigned into four groups according to the season of the year of birth (n = 4 in each group). At 75 days of age at the initiation of the study, they weighed between 3.2 and 5.1 kg (4.24 ± 0.53 kg). The onset of ovulatory activity was determined by PGS and IGF-I serum concentrations every 10 days starting from 75 days to 155 days of age. The onset of puberty in CDG kids occurred on average at 141.15 ± 2.66 days of age, but varied depending on the season of birth. All CDG kids born during summer were in heat by 155 days of age. In the other groups (kids born in autumn, winter or spring), one goat in each group was not in heat. Changes in blood serum PGS and IGF-I concentrations during prepubertal and pubertal periods could aid in the evaluation of reproductive status and determination of the onset of puberty in CDG during all seasons of the year in a moderate climate region.

Comparison of Intrauterine Antibiotics versus Ozone Medical Use in Sheep with Retained Placenta and Following Obstetric Assistance.

This study outlines a new approach to reproductive tract treatment using ozone foam spray for certain ovine obstetrical problems, such as retained foetal membranes and possible uterine infections following obstetric assistance (OA), in comparison with classical antibiotics treatments. The study was conducted on 256 ewes from 11 sheep farms in north-western Croatia. A total of 139 ewes were diagnosed with dystocia (DT) and 49 with retention of placenta (RP). Ewes with RP were treated either with ozone foam spray (Riger spray G; Novagen(®) ) applied into the body of the uterus for 2-3 s (first or RPO group; n = 24) or with two foaming, intrauterine tablets of oxytetracycline hydrochloride (Geomycin(®) F) (second or RPA group; n = 25). The third and fourth groups consisted of ewes that received OA for dystocia (including ringwomb, foetal oversize and assistance of abnormal position and posture). The third group (DTO; n = 70) was treated with ozone foam spray, while ewes in fourth group (DTA; n = 69) were treated with antibiotics. The ewes in the control group (CTL) with physiological puerperium were randomly selected (n = 70) from all herds. Transrectal ultrasonography (transversal diameter of uterine horns) was used for the control of uterus regression on days 2 and 25 after parturition. There was a difference in transversal uterine horn diameter in the RP groups, that is RPO and RPA (5.40 ± 0.53 cm vs. 5.43 ± 0.40 cm), ewes with dystocia, that is DTO and DTA (5.37 ± 0.49 cm vs. 5.54 ± 0.60 cm) and ewes from the CTL group (4.98 ± 0.35 cm) one day after parturition. Average transversal uterine diameter of all groups at day 25 post-partum was 1.80 ± 0.15 cm. The intrauterine ozone treatment in ewes with RP and after manual obstetrics attained similar results to spontaneously delivered ewes (CTL group), showed as the physiological regression of the uterus with a similar transversal diameter without the presence of lochia in the uterine lumen, indicating that this could be a novel potential alternative therapy.

Influence of Body Condition on Serum Metabolic Indicators of Lipid Mobilization and Oxidative Stress in Dairy Cows During the Transition Period.

The objectives of this study were to examine the influence of body condition of cows on metabolic and antioxidative status, as well as to investigate the relationship between metabolic indicators of lipid mobilization and oxidative stress during transition period. The study was conducted on 24 Holstein-Friesian dairy cows divided into 2 groups according to their body condition score (BCS) as optimal (n = 12; BCS from 3.25 to 3.75) or adipose (n = 12; BCS ≥4). Metabolic status (glucose, triglycerides, total cholesterol, HDL cholesterol, NEFA and BHB), paraoxonase-1 (PON1) and apolipoprotein A-I (ApoA-I) were analysed in sera taken on days -30, -10, -2, 0, 5, 12, 19, 26 and 60 relative to parturition. Adipose cows had significantly higher glucose concentration at parturition being significantly decreased after parturition on days 12 and 19. Total cholesterol and HDL-C concentrations were the lowest at parturition and significantly higher on days 26 and 60 after parturition in both groups of cows. Both investigated groups had significantly higher NEFA concentration from parturition until day 19 after parturition, indicating energy deficit and an increased lipid mobilization after calving. There were no significant differences in BHB concentration during transition period in both groups. No significant differences were found in PON1 activity and ApoA-I concentration during transition period in both groups of cows. However, in adipose cows, although not significantly different, PON1 was decreased from calving until day 19 after parturition indicating a disturbance in antioxidative status in adipose cows. PON1 significantly positively correlated with total cholesterol and HDL-C concentrations and negatively with NEFA indicating a strong relationship of PON1 with lipid metabolism. Significant positive correlation between NEFA and BHB in both groups of cows points out on energy deficit during transition period that cows tend to overcome by lipid mobilization providing alternative source of energy needed for parturition and lactation.

The intrauterine treatment of the retained foetal membrane in dairy goats by ozone: novel alternative to antibiotic therapy.

One of the major post-parturient complications in dairy goats is the retention of foetal membrane (RFM), which negatively influences their health, reproductive efficacy and welfare. The aim of this study was to compare the efficiency of intrauterine either ozone (OZ) or antibiotic (AB) treatments to establish the use of OZ as a novel and potential alternative to AB therapy in does with the RFM. The study was performed on 7 herds of dairy goats (n = 563) kept in the farms in Croatia. The conception rate was 563 of 641 total matings or 87.83%. The does from selected farms were observed during early puerperium and were divided into animals without the RFM (n = 522) and with the RFM (n = 41), treated either with foam spray OZ (n = 21) or with foaming AB oxytetracycline tablets (n = 20). The does with the RFM were mated successfully and became pregnant next kidding season, regardless of the treatment applied. Treatment with OZ attained similar results to the standard AB therapy, indicating that it could be novel potential alternative therapy of the RFM in dairy goats.

Secretion of immunomodulating neuropeptides (VIP, SP) and nitric oxide synthase in porcine small intestine during postnatal development.

Immunohistological identification/localization of immunomodulating neuropeptides [vasoactive intestinal polypeptide (VIP) and substance P (SP)] and enzyme nitric oxide synthase (NOS) as well as histomorphometric analyses of kinetics of their release and development of respective nerve fibers density during postnatal ontogenesis of porcine intestinal mucosal immune system (IMIS), were performed in order to assess the role of these molecules involved in maturation of the IMIS. The kinetcs of reactions to VIP, SP and NOS were demonstrated in the samples of jejunum and ileum from conventionally reared pigs. The samples were obtained at 0, 7, 14, 21, 28, 35, 42 and 49 days of age and processed for immunohistological staining. The VIP+ reaction was prevalently visible in the epithelial layer, lamina propria and Lieberkühn crypts (Lc) but also in the submucosa and lamina muscularis along blood and lymphatic vessels. The SP+ fibers were regularily distributed along enteric neurons in the muscular layer. The reaction to NOS was demonstrated in both mucosa and submucosa of ileum and jejunum and in the ileal Peyer's patches (PP). Intensity of the reaction was more pronounced in the epithelial layer and numerous NOS+ cells were observed around the Lc and inside the follicles of the PP. Also, we have noticed NOS+ blood vessels, particular neurons and nerve fibers in the submucosa and muscular layer of the small intestine. By analyzing quantitative patterns of SP+, VIP+ fibers and release of NOS we have concluded that intensity of their reactions gradually increases with age, except a short period of stagnation after weaning (at age of 28 days), reaching the highest values in the pigs aged between 42 and 49 days. The values obtained by Sperman rank order correlation test (rs) between days of age of pigs and intensity of the reactions in their jejunum/ileum to VIP (rs=0.97/0.95), SP (rs=0.97/0.97) and NOS (rs=0.98/0.95), respectively, showed positive correlations (P<0.05) according to Roemer Orphal scale. Current study showed that postnatal development of porcine IMIS was accompanied by a substantial increase in the secretion of neuropeptides/enzyme tested and that these molecules may participate in the functional maturation of immunoregulatory/bactericidal mechanisms of the local (intestinal) immune defense in young pigs.

Histomorphometric evaluation of intestinal cellular immune responses in pigs immunized with live oral F4ac+ non-enterotoxigenic E. coli vaccine against postweaning colibacillosis.

Enterotoxigenic Escherichia coli (ETEC) infection is the most common type of porcine postweaning colibacillosis (PWC). Among fimbriae of porcine ETEC strains the best studied family of fimbriae are the members of F4 adhesins, existing in at least three variants: ab, ac, ad. Active immunization against porcine PWC is difficult due to: i) ETEC strains are only one of the essential predisposing factors, ii) the success of vaccinal antigen uptake depends on the presence of enterocyte receptors for F4 adhesins, iii) the intestinal immune system may react with tolerance or hypersensitivity to the same antigens depending on the dose and form of the vaccinal immunogen, and iv) kinetics of the specific immune responses may be different in the case of F4 (earlier) and the other ETEC adhesins, particularly F18 (later). The aim of this study was to test the effectiveness of a live attenuated F4ac+ non-ETEC vaccine against porcine PWC by analyzing quantitative differences in the small intestinal lymphoid and myeloid cell subsets of immunized (with or without levamisole given as an adjuvant) vs control non-immunized pigs. Four week-old pigs were intragastrically immunized with a vaccine candidate F4ac+ non-ETEC strain 2407 at day 0, challenged 7 days later with a virulent F4ac+ strain ETEC 11-800/1/94, euthanatized at day 13 and sampled for immunohistology. Non-immunized pigs received saline at day 0 and were processed as the principals. Immuno-phenotypes of lymphoid and myeloid cell subsets were demonstrated within jejunal and ileal mucosa by immunohistochemical avidin-biotin complex method and corresponding morphometric data were analyzed using software program Lucia G for digital image analyses. Monoclonal antibodies reactive with surface molecules on porcine immune cells such as CD3, CD45RA, CD45RC, CD21 and SWC3 enabled clear insight into distribution patterns and amount of these cells within the gut-associated lymphoid tissues (GALT) examined. The numbers of jejunal and ileal cell subsets tested were significantly increased (at P<0.5 or lower) in both principal groups (vaccinated or levamisole primed-vaccinated) of pigs, compared to those recorded in the control non-vaccinated pigs. Based on the histomorphometric quantification of porcine intestinal immune cells from the GALT compartments tested, it is possible to differentiate the responses of pigs immunized by an experimental mucosal vaccine from those of non-immunized pigs.

Increased number of intestinal villous M cells in levamisole -pretreated weaned pigs experimentally infected with F4ac⁺ enterotoxigenic Escherichia coli strain.

Immunoprophylaxis of porcine postweaning colibacillosis (PWC) caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbriae is an unsolved problem. Just as ETEC strains can exploit intestinal microfold (M) cells as the entry portal for infection, their high transcytotic ability make them an attractive target for mucosally delivered vaccines, adjuvants and therapeutics. We have developed a model of parenteral/oral immunization of 4-weeks-old pigs with either levamisole or vaccine candidate F4ac(+) non-ETEC strain to study their effects on de novo differentiation of antigen-sampling M cells. Identification, localization and morphometric quantification of cytokeratin 18 positive M cells in the ileal mucosa of 6-weeks-old pigs revealed that they were: 1) exclusively located within villous epithelial layer, 2) significantly numerous (P< 0.01) in levamisole pretreated/challenged pigs, and 3) only slightly, but not significantly numerous in vaccinated/challenged pigs compared with non-pretreated/challenged control pigs. The fact that levamisole may affect the M cells frequency by increasing their numbers, makes it an interesting adjuvant to study development of an effective M cell-targeted vaccine against porcine PWC.

Histomorphometric characteristics of immune cells in small intestine of pigs perorally immunized with vaccine candidate F18ac nonenterotoxigenic E. coli strain.

Colidiarrhea and colienterotoxemia caused by F4(+) and/or F18(+) enterotoxigenic E. coli (ETEC) strains are the most prevalent infections of suckling and weaned pigs. Here we tested the immunogenicity and protective effectiveness of attenuated F18ac(+) non-ETEC vaccine candidate strain against challenge infection with F4ac(+) ETEC strain by quantitative phenotypic analysis of small intestinal leukocyte subsets in weaned pigs.We also evaluated levamisole as an immune response modifier (IRM) and its adjuvanticity when given in the combination with the experimental vaccine. The pigs were parenterally immunized with either levamisole (at days -2, -1 and 0) or with levamisole and perorally given F18ac(+) non-ETEC strain (at day 0), and challenged with F4ac(+) ETEC strain 7 days later.At day 13 the pigs were euthanatized and sampled for immunohistological/histomorphometrical analyses. Lymphoid CD3(+), CD45RA(+), CD45RC(+), CD21(+), IgA(+) and myeloid SWC3(+) cell subsets were identified in jejunal and ileal epithelium, lamina propria and Peyer's patches using the avidin-biotin complex method, and their numbers were determined by computer-assisted histomorphometry. Quantitative immunophenotypic analyses showed that levamisole treated pigs had highly increased numbers of jejunal CD3(+), CD45RC(+) and SWC3(+) cells (p<0.05) as compared to those recorded in nontreated control pigs.In the ileum of these pigs we have recorded that only CD21(+) cells were significantly increased (p<0.01). The pigs that were treated with levamisole adjuvanted experimental vaccine had significantly increased numbers of all tested cell subsets in both segments of the small intestine. It was concluded that levamisole adjuvanted F18ac(+) non-ETEC vaccine was a requirement for the elicitation of protective gut immunity in this model; nonspecific immunization with levamisole was less effective, but confirmed its potential as an IRM.