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In this study, the effects of melatonin treatment on growth, redox status and immunity in prenatally stressed newborn lambs were evaluated. Thirty-seven newborn lambs were allocated into two groups (melatonin-MEL and control-CON), based on whether their mothers were treated with melatonin implants or not, respectively. All pregnant ewes were exposed to heat stress. The body weight of lambs was recorded at birth (L0), and then on days 15 (L15) and 40 (L40). Redox biomarkers [total antioxidant capacity (TAC), glutathione (GSH), thiobarbituric acid reactive substances (TBARS)] were assayed in blood samples collected from lambs on days L0, L1, L2, L5, L10 and L40. Chemical analysis and antioxidant capacity were evaluated in colostrum and milk samples collected at the same time points with blood samples. Cytokines (IL-1β, IL-6, IL-10, IFN-γ) and immunoglobulin (IgG) were assayed in blood and colostrum samples collected from ewes on days L0 and L1, and in lambs' blood on days L0, L1 and L2. The results revealed that body weight gain of newborn lambs did not differ between the two groups ( p > 0.05). Better redox status was found in MEL lambs until L2, as well as higher antioxidant capacity in the colostrum of MEL ewes compared to CON ones on day L0 ( p < 0.05). In MEL ewes' colostrum, higher protein content was measured on day L0 and higher fat content on L1 compared to CON group ( p < 0.05). The highest level of IL-6 was found in MEL ewes on L1, with a concomitant increase of IL-10 level in MEL lambs in comparison to CON lambs on L2. Moreover, CON colostrum resulted in a higher level of IL-10 within time, coupled with an increased level of IgG found in lambs' plasma on L2 ( p = 0.04). This study indicated that melatonin could be administered as antioxidant and immune-modulatory regime in prenatally stressed offspring in order to cope with the crucial first days of their life. This effect of melatonin was also amplified by crosstalk between IL-6, IL-10 and IgG production, resulting in an improved quality of produced milk.

Heat stress is a known promoter of reactive oxygen species generation, which may compromise pregnancy and foetal development. Melatonin is a pleiotropic molecule that regulates various processes including pregnancy. Thus, it could be used to ameliorate the redox status of pregnant heat-stressed ewes and the outcome of their pregnancy. Sixty-eight ewes participated in the study, which were allocated into two equal groups, i.e., Melatonin (M) and Control (C) group. All ewes were exposed to heat stress from D0 to D120. In both groups, after oestrus synchronization of ewes, rams were introduced to them for mating (D16). In M group, starting with sponges' insertion (D0), melatonin implants were administered four-fold every 40 days. Pregnancy diagnosis was performed by means of ultrasonography. Daily evaluation of temperature humidity index (THI), rectal temperature, and breathing rate were performed throughout the study. Blood samples were collected repeatedly from D0 until weaning for assaying redox biomarkers. Milk yield was measured thrice during puerperium. The results showed that melatonin administration throughout pregnancy improved the redox status of heat-stressed ewes and increased the mean number and bodyweight of lambs born per ewe, as well as the milk production. Therefore, melatonin may be used as antioxidant regimen in heat-stressed ewes for improving their reproductive traits., Competing Interests: The authors declare no conflict of interest.

The purpose of this study was to investigate whether fertile or non-fertile inseminations (AI) in synchronized ewes are correlated with the electrical resistance of cervical mucus (ERCM) and the ovarian steroid concentration. AIs were performed either at fixed-time (group A) or after estrus detection (group B). Retrospective analysis revealed that at AI, pregnant ewes had lower ERCM values and progesterone concentrations than non-pregnant ones (p<0.05). It appears that ERCM may be used as an additional index for fertility enhancement of inseminated ewes., (Copyright © 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Blood concentrations of progesterone, FSH and oestradiol in Karagouniko ewes subjected to laparoscopic ovum pick-up (OPU) at specific stages of induced oestrous cycle, were measured. Twenty-four cyclic ewes were randomly allocated into four equal groups (A, B, C and D). Oestrus was synchronized with progestagen intravaginal sponges and detected by teaser rams (oestrus: day 0). In group A, during the induced oestrous cycle, OPU was performed on days 4, 9 and 14 (sessions A1, A2 and A3, respectively). In group B and group D, OPU was performed once, on day 9 and 14, respectively. In group C (controls), endoscopic observation of follicular population was performed three times, as in group A. Starting at sponge removal, progesterone was measured in blood samples collected on 22 daily occasions and oestradiol in samples collected on 27 occasions collected at various time-points starting 2h before to 24h after OPU. Follicular populations did not differ among A1, A2, A3 or between C1, C2, C3 and A1, A2, A3 or A1, B, D, respectively. Oocytes of better quality (category '1' or '2') were collected at A3 session compared with A1 (P<0.05). Progesterone concentration and oestrous cycle length did not differ among groups. Decreased oestradiol concentrations followed by FSH increase were recorded 3-5h after OPU. The results confirm the regulatory role of oestradiol on FSH secretion. The quality of collected oocytes was improved in subsequent pick-up sessions in the oestrous cycle. Moreover, OPU at specific stages of the luteal phase of the cycle, even when applied repeatedly, do not affect the normal oestrous cycle length of ewes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Two consecutive randomized double-blind field fertility experiments were conducted over a 4-month period and aimed at evaluating the association of two commercial soybean lecithin-based extenders (AndroMed [Minitub, Tiefenbach, Germany] and BioXcell [IMV Technologies, L'Aigle, France]) with pregnancy rates of chilled-stored (CS) and frozen-thawed (FT) ram semen. Semen samples with more than 2 × 10(9) sperm per mL and 70% progressive motile spermatozoa were collected via an artificial vagina from twelve proven fertile Chios rams, split-diluted with the above mentioned extenders, packaged in 0.25 mL straws and either stored at 5 ± 1 °C for 30 to 36 hours or frozen and thawed. Non-lactating multiparous ewes were inseminated in progestagen-synchronized estrus either with CS (AndroMed: N = 212 and BioXcell: N = 206; intracervical AI) or with FT (AndroMed: N = 114 and BioXcell: N = 92; laparoscopic intrauterine AI) semen. Ovulation was confirmed in all ewes based on determination of blood plasma progesterone (>1 ng/mL) 8 days post AI. Ewes were screened for pregnancy diagnosis by transabdominal ultrasonography 65 days post AI. BioXcell was superior to AndroMed in preserving the fertilizing potential of CS (P < 0.05) and FT (P < 0.005) semen. In AndroMed-stored semen, young rams (1.5-2.5 years old, N = 8) had a pregnancy rate (59.1%; 124/210) lower than that (72.4%; 84/116) of mature rams (4.5 to 5.5 years, N = 4; P < 0.025). Compared with AndroMed extender, processing of young ram semen in BioXcell extender improved pregnancy rates of CS (66.7%; 88/132 vs. 83.9%; 94/112; P < 0.005) and FT (46.2%; 36/78 vs. 71.0%; 44/62; P < 0.01) spermatozoa. Both extenders were similarly effective in preserving pregnancy rates of mature ram semen (P > 0.05). Ram-by-extender interactions were significant for pregnancy rates of CS and FT semen. Irrespective of extenders, overall pregnancy rates after intracervical and intrauterine AI were 75.1% and 62.2%, respectively (P < 0.001). In conclusion, BioXcell is a suitable extender for short- and long-term storage of ram semen. Selection of the ewes, farms, and extenders for intracervical AI programs can contribute to satisfactory fertility rates with semen preserved more than 24 hours at 5 °C., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The pharmacokinetics of amoxicillin (AMX) in blood serum (SBS) and tissue cage fluid (TCF) was studied in sheep. Four tissue cages, prepared from silicone rubber tubing, were subcutaneously inserted in the neck area (two on each side) of the experimental animals and AMX was administered both intravenously (IV) and intramuscularly (IM) at the dose rate of 15mg/kg bodyweight. The impact of local inflammation on AMX distribution in TCF was studied after intra-cavity injection of a lambda carrageenan solution in one of the two tissue cages used after each administration. In contrast to the three-compartment AMX disposition after IV injection, two-compartment, absorption-limited pharmacokinetics was observed after IM administration. Non-inflamed and inflamed TCF data revealed, in all cases, the attainment of low, but prolonged concentrations and absence of an inflammation-induced effect on AMX penetration into and elimination from TCF., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The pharmacokinetics of amoxicillin (AMX) were investigated in sheep following intravenous (i.v.) and intramuscular (i.m) injection, comparing two different drug formulations, a conventional and a long-acting AMX-trihydrate suspension. For the i.m. application two different injections sites, the neck area and the hind limb were used to identify possible differences in the kinetic parameters related to the site of injection. A three-compartment open model could best describe AMX disposition after i.v. administration. Data analysis after i.m. administration of the conventional suspension at both injection sites revealed the occurrence of a flip-flop phenomenon, clearly indicating that absorption of AMX is the rate-limiting step of its overall disposition. A moderate effect of the injection site was observed with a tendency for the neck area to be advantageous, mainly in terms of rate rather than extent of absorption. Injection of the long-acting formulation led to a focal depot formation, thus yielding lower but remarkably prolonged serum AMX levels reflected in the respective terminal half-lives. The concentration-time profile of AMX after administration of the long-acting formulation was less affected by the injection site, but the low serum levels justify its use only in cases in which a high susceptibility of the involved bacterial population is confirmed.

Purpose: In this study we evaluated the clinical usefulness of serum pro-I collagen peptide (PICP) and I collagen telopeptide (ICTP) as indicators of early bone metastases in patients with breast (BC), lung (LC), urinary bladder (UBC) and prostate cancer (PC)., Patients and Methods: 305 patients were examined. 145 had histologically confirmed BC (92 with bone metastases), 20 UBC (6 with bone metastases), 11 LC (3 with bone metastases) and 129 PC (68 with bone metastases). In BC patients we compared the PICP and ICTP levels with those of CA 15-3, CEA and bone scintigraphy. Patients with LC and UBC had PICP and ICTP measurements, PC patients had serum PICP, prostate specific antigen (PSA) measurements and bone scans. 104 healthy individuals served as controls., Results: ICTP and CA 15-3 levels were significantly higher in patients with BC and bone metastases in comparison to patients without metastases (p <0.05), while PICP and CEA were only marginally higher. Significant correlation was observed between existence of bone metastases and ICTP levels (p <0.05). The sensitivity of PICP, ICTP, CEA and CA 15-3 was 28.1, 48.6, 42, and 78%, respectively and specificity was 83.9, 94, 65 and 86%, respectively. ICTP and CA 15-3 were the most reliable markers for early diagnosis of bone metastases in BC. PICP alone or with ICTP were not sensitive enough. Only CA 15-3 showed sensitivity 78% and specificity 86%. When combined CA 15-3, ICTP and CEA the sensitivity and specificity increased to 82% and 96%, respectively. Furthermore, PICP and PSA levels were significantly higher in patients with PC and bone metastases in comparison to patients with benign prostate hyperplasia (BPH) (p <0.0001) or in patients with PC without bone metastases (p <0.0005 for PICP and p <0.0001 for PSA). The co-evaluation of PICP and PSA improved the sensitivity (78%), specificity (96%), accuracy (97%) and positive predictive value (97%). In LC patients, ICTP levels differed significantly between patients with and without bone metastases (p=0.025). In UBC patients, PICP levels differed significantly between patients with and without bone metastases (p=0.017)., Conclusion: ICTP and CA 15-3 are the most reliable markers for early diagnosis of bone metastases in BC patients. PICP could be useful for diagnosing early bone metastases of PC and combined with PSA and bone scan can be an additional tool in the follow-up of PC patients. For LC patients, ICTP showed a significant difference in the discrimination of patients with and without bone metastases. In UBC patients, PICP showed a significant difference in the discrimination of patients with and without bone metastases.

The effect of porcine or ovine FSH on the maturation rate of porcine oocytes and on the time course of meiotic progression was studied. Groups of 20 grade-A cumulus oocyte complexes, aspirated from slaughterhouse cycling-gilt ovaries, were cultured in vitro in 400 mul of Modified Parker's Medium supplemented with oestrous cow serum and porcine FSH (Folltropin(R)-V, 0.50 mg/ml) or ovine FSH (Ovagen(TM), 0.44 iu/ml), in four-well dishes under mineral oil, at 38.5 degrees C, 5% CO(2) in humidified air. At the end of each 3-h interval, from 3 to 42 h of culture, the nuclear status of oocytes was assessed microscopically (1000x), after fixation (methanol/acetic acid: 3/1) and orcein (2%) staining. Oocytes were classified as (i) immature (IMM), i.e. oocytes at germinal vesicle stage, germinal vesicle break down and prophase I, (ii) metaphase I (MI) and (iii) metaphase II (MII), i.e. oocytes at anaphase I, telophase I and metaphase II. Data were analysed using regression analysis, chi-square and t-test. Nuclear status was assessed in 1610 oocytes (porcine FSH: 787, ovine FSH: 823). Most of the oocytes were at MI from 24 to 33 h (porcine FSH 60.27%, ovine FSH 42.80%, p < 0.001) and at MII from 36 to 42 h (porcine FSH 80.38%, ovine FSH 67.45%, p < 0.01) of culture. Significantly higher maturation rate was observed in porcine FSH than in ovine FSH treated oocytes (86.69 +/- 12.97%, 71.34 +/- 9.86%, mean +/- SD, p < 0.05), after 42 h of culture. In conclusion, under the specific culture conditions, porcine FSH seems to support pig oocyte maturation better than ovine FSH.

A novel method for oestrus-ovulation synchronization in sheep followed by fixed time insemination is presented herewith. Mature dry ewes (n = 28) of Karagouniko breed being at an unknown stage of the oestrous cycle, were used during the middle of breeding season. The treatment protocol consisted of an initial administration of a GnRH analogue followed 5 days later by a prostaglandin F2alpha injection. Thirty-six hours later a second GnRH injection was administered to synchronize ovulation, and laparoscopic intrauterine insemination was performed 12-14 h later. Three days after insemination, fertile rams were introduced into the flock twice daily and oestrus-mating detection was carried out. For progesterone (P(4)) determination, blood samples were collected on alternate days, starting 2 days before the first GnRH injection and continuing for 17 days after insemination. An additional sample was taken on the day of insemination. Pregnancy diagnosis was carried out by trans-abdominal ultrasonography. Fourteen ewes (50%) conceived at insemination and maintained pregnancy; from the remainder 14 ewes 10 became pregnant at natural service, while four, although they mated at least two to three times, failed to conceive. In response to the first GnRH, P(4) concentration increased at higher levels in ewes that conceived at AI compared with those that failed to conceive (47.54 and 22.44%, respectively; p < 0.05). Significant differences (p < 0.05) in mean P(4) concentration between pregnant and non-pregnant animals were detected 1 day before AI (0.17 +/- 0.06 and 0.26 +/- 0.14 ng/ml, respectively) on the day of AI (0.15 +/- 0.04 and 0.24 +/- 0.08 ng/ml, respectively) as well as 9 and 11 days thereafter (0.48 +/- 0.12 and 0.38 +/- 0.12 ng/ml; 0.68 +/- 0.14 and 0.50 +/- 0.18 ng/ml, respectively). These results indicate that using the proposed protocol, an acceptable conception rate can be achieved which could be further improved by modifying the time intervals between interventions.

A new analytical method for the simultaneous quantitative determination of albendazole metabolites in sheep spermatozoa and seminal plasma at levels down to 46.5 ng/mL for albendazole sulphoxide (ABZ-SO), 7.5 ng/mL for albendazole sulphone (ABZ-SO2) and 12 ng/mL for albendazole 2-aminosulphone (ABZ-SO2NH2) has been developed. Analytes were extracted from alkalinized samples with ethyl acetate. Separation was carried out on a C18 column in the presence of tetra-n-butylammonium (TBA) hydrogen sulphate and octanesulphonate sodium (OCT), as ion-pair agents. Fluorometric detection was performed with excitation and emission wavelengths set at 290 and 320 nm, respectively. Accuracy data showed overall recoveries (+/-S.E.M.) of 83.1+/-1.2% for ABZ-SO, 98.8+/-0.6% for ABZ-SO2 and 85.3+/-0.7% for ABZ-SO2NH2, in spermatozoa. Respective values in seminal plasma were 88.0+/-0.9%, 97.7+/-0.5% and 93.2+/-1.7%. Precision data suggested coefficient of variation (CV%) values lower than 5.9% for spermatozoa and 3.8% for seminal plasma. The method was successfully applied for the determination of the three albendazole metabolites in semen samples collected from rams that had been orally administered albendazole.

Synchronisation of oestrus in Karagouniki ewes by administration of the standard dose of progesterone results in lower fertility than observed when these ewes ovulate naturally. This suggests that the optimum dose of progesterone may be breed dependent. The exogenous progesterone may perturb the concentrations of oestradiol-17beta and progesterone in blood plasma and the oviductal wall. This possibility was investigated using Karagouniki ewes allocated at random to three treatments (n=4 per treatment). Ewes were allowed to exhibit natural oestrus (N) or oestrus was synchronised by administration of 250 mg (LP) or 375 mg (HP) progesterone (subcutaneous implants) followed by PMSG at 8 mg/kg live weight i.m. 14 days later. Oestrus was observed using teaser rams. Blood samples were collected for plasma oestradiol-17beta and progesterone assay from the onset to the end of oestrus at 2 h intervals. The uterus of each ewe was recovered at the end of oestrus and samples of the oviductal wall were taken from both oviducts and prepared, separately, for progesterone and oestradiol-17beta assay. Statistical analysis was performed using univariate analysis of variance. Plasma oestradiol-17beta concentrations from the onset to the end of oestrus were highest for N ewes and lowest for HP ewes with the values for LP ewes occupying an intermediate position. The differences were significant (P<0.05) between HP and the other two treatments from 4 to 12 h after the onset of oestrus and then between all treatments until the end of oestrus. Plasma progesterone levels were similar and fairly constant from the onset to the end of oestrus for N and LP. The plasma progesterone levels for HP were significantly (P<0.05) higher than for the other two treatments throughout oestrus. In oviductal wall samples, the oestradiol-17beta concentration was significantly (P<0.05) higher for N ewes than for synchronised ewes and the levels were similar for LP and HP ewes. The concentration of oestradiol-17beta differed (P<0.05) between right and left oviducts for N ewes but not for ewes of either of the synchronised oestrus treatments. Progesterone concentrations in oviductal wall samples were highest (P<0.05) for HP ewes and the values for N and LP ewes were similar. The concentration of progesterone did not differ between right and left oviductal wall samples within treatments. It was concluded that the higher dose of exogenous progesterone perturbed the levels of oestradiol-17beta and progesterone in blood plasma and the oviductal wall, and this could explain the lower levels of fertility (relative to naturally occurring oestrus) observed when this protocol is used for Karagouniki ewes in practice.

Organochlorine pesticides and polychlorinated biphenyls are widely used in agriculture and industry, respectively, and may affect male reproduction function. Although several pollutants have been detected in human semen, similar studies in farm animals have not appeared. In the present study, the semen of bulls, rams, goats, and boars was assayed for the organochlorine pesticides hexachlorobenzene (HCB), hexachlorocyclohexane (HCH, alpha-, beta-, and gamma-isomers), dieldrin, and heptachlor epoxide, for DDT-related chemicals (o,p'-DDE, p,p'-DDE, p,p'-DDD, p,p'-DDT), and for the PCBs congeners (PCB-52, -101, -138, -150, and -180). In all species of farm animals, the most frequently detected pollutants were p,p'-DDE (80-100% of samples), HCB (73.9-100%), and gamma-HCH (69.6-100%). Species differences in the concentrations of HCB, alpha-, beta-, and gamma-HCH, dieldrin, p,p'-DDE, p,p'-DDD, and PCBs were noted as well as differences in the concentrations of some isomers of HCH, DDT-related chemicals, and PCB congeners in the same species.

Organochlorine pesticides and polychlorinated biphenyls are widely used in agriculture and industry, respectively. The present study assessed the burden of environmental pollutants in the follicular fluid of farm animals (cattle, sheep, goats, and pigs). An analytical method combining a solid-phase extraction with (C(18)) for clean-up and GC-electron capture detection using a capillary column was implemented for isolation and determination of organochlorine pesticides and polychlorinated biphenyls (PCBs). Of the organochlorine pesticides, hexachlorobenzene (HCB), hexachlorocyclohexane (HCH, alpha-, beta-, and gamma-isomers), dieldrin, heptachlor epoxide, and the DDT-related chemicals (o,p'-DDE, p,p'-DDE, p,p'-DDD, p,p'-DDT) were detected and of the PCBs, the congeners PCB-52, -101, -138, -153, and -180 were detected. In all species of farm animals, the most frequently detected pollutant was gamma-HCH (90-100% of samples) followed by HCB (80-100%), and p,p'-DDE (75-90.91%). Species differences in the concentrations of HCB, beta-HCH, heptachlor epoxide, and DDT-related chemicals in follicular fluid were noted as well as differences in the concentrations of some pollutants within the same species.

In a series of consecutive blood sampling in 15 days intervals over 15 weeks after implantation of melatonin in rams an increased mean value, basal level and number of peaks of testosterone was observed in samples of the third fortnight (45th day). This increase was greater in the autumn (breeding season) than in spring (non-breeding season). Total acrosin activity in spermatozoa was increased between days 35-56 (autumn) and days 49-70 (spring) after implantation and the relative increase was higher in autumn than in spring. The increase of acrosin activity was independent of the changes of testosterone. An increase of acrosin activity by melatonin, in cases of low activity, might improve fertilization rates in sheep not only during the breeding season, but also during the non-breeding season (after oestrus induction).

Some parameters of haem synthesis were estimated in 60 uraemic patients (30 non-dialysed, 30 dialysed) and in 30 matched controls. Serum delta-aminolaevulinic acid and erythrocyte coproporphyrin and protoprophyrin were found significantly higher in the non-dialysed uraemics than in the controls. Erythrocyte delta-aminolaevulinic acid dehydrase (ALA-D) activity was 498 +/- 174 mumol/h.l in the non-dialysed patients, 321 +/- 146 in the dialysed (just before haemodialysis) and 833 +/- 281 in the healthy controls, the differences between these groups all being statistically significant (p less than 0.001). After haemodialysis the enzymic activity in the dialysed group increased significantly (380 +/- 167, p less than 0.001), but remained lower than normal (p less than 0.001). A similar pattern - although with less statistical significance of the differences between groups - was observed concerning erythrocyte uroporphyrinogen I synthase activity. Incubation of normal erythrocytes with uraemic plasma resulted in a considerable decrease of their ALA-D activity (from 830 +/- 263 to 616 +/- 126) while incubation of uraemic erythrocytes with normal plasma increased their ALA-D (from 384 +/- 139 to 494 +/- 77). Addition of zinc in the haemolysate caused a similar induction of ALA-D in both controls and uraemics. The zinc-induced uraemic ALA-D practically reached normal levels. The mechanism of enzymic depression and the possible role of elevated delta-aminolaevulinic acid concentrations (to which depressed ALA-D activity considerably contributes) in the pathogenesis of the neurologic manifestations of uraemia, are discussed.