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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&rsquo, 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.

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 &gt, 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 &lt, 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 &lt, 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.

The effect of prolonged aflatoxin B1 (AFB1) administration on blood serum oestradiol-17β and progesterone concentrations in goats during the luteal phase and the synchronized oestrus was investigated. Thirty-six Greek indigenous primiparous goats were used, during the oestrus period; 12 goats received, per os, 50 μg (treated group T50) and 12 goats received 100 μg (treated group T100) AFB1/day/head, respectively, for approximately 1.5 month, while 12 goats served as controls (C). On day 36 of the experiment, each goat was injected, i.m, 0.5 ml prostaglandin F2α (PGF2α). Blood samples were collected from each goat twice a week, before PGF2α injection, as well as every 4 hours from the onset to the end of the synchronized oestrus. Oestradiol-17β and progesterone concentrations in blood serum were determined using radioimmunoassay. During the whole luteal(s) phase(s), linear regression analysis revealed a significant negative dependence (P 0.05). In conclusion, prolonged AFB1 administration at doses of 100 or even of 50 μg/day/head changes the hormonal pattern in blood during the luteal phase and the synchronized oestrus of goats, being in oestrus period.

The aim of this study was to investigate a) the effect of equine Chorionic Gonadotropin (eCG), in the dosage used for oestrus synchronization or for superovulation, on the activity of α-mannosidase (α-MAN) and β-N-acetyloglucosaminidase (β-NAGASE) in the genital tract of ewes before maternal recognition of pregnancy and b) the possible relationships among ovarian steroids concentration, glycosidase activity and embryo yield and quality. Twenty-four Chios ewes were treated with progestagen intravaginal sponges for 12 days and eCG [superovulation (SOV), n = 7, 1000 IU; oestrus synchronization (OS), n = 7, 500 IU; controls (C), n = 10, 0 IU] at sponge removal. Ovarian response was assessed and embryos were collected on day 6 of the oestrous cycle, after slaughter. Oestradiol-17β and progesterone concentrations were determined in blood serum samples collected before slaughter using a radioimmunoassay. The activities of α-MAN and β-NAGASE were assayed, photometrically, in uterine horn flushing (UHF) samples, caruncular endometrium (CE) and intercaruncular (ICE) endometrium samples, collected after slaughter. Alpha-MAN activity was higher in the UHF, CE and ICE of SOV ewes, as well as in the UHF and ICE of OS ewes, compared to controls. Beta-NAGASE activity in the UHF and in the ICE of SOV ewes was lower than in C ewes. Alpha-MAN activity in the UHF was positively correlated with the response to superovulation in terms of total recovered structures, embryos or high-quality embryos collected from the ewes. Beta-NAGASE activity in the CE correlated to the number of corpora lutea observed on the ovaries, at the day of embryo collection. Beta-NAGASE activity in UHF and α-MAN activity in the ICE were negatively related to oestradiol-17β concentration in blood serum; β-NAGASE activity in UHF and in ICE was negatively related to progesterone concentration in blood serum. In conclusion, eCG in the dosage used for superovulation and, in a lesser degree, in the dosage used for oestrus synchronization affects glycosidase activity in the genital tract of Chios breed ewes, before maternal recognition of pregnancy; glycosidase activity in the genital tract correlate to ovarian response and embryo yield. Further research is necessary to fully clarify the interrelationship between ovarian steroids concentration and glycosidase activity in the genital tract of ewes after hormonal treatment.

Epidemiological studies of disease ecology typically ignore the influence of host sex, age, nutritional condition and immunocompetence factors. However, this can lead to shortcomings and incorrect conclusions regarding the mechanisms of pathogen transmission and prevalence in wild animals. In many European countries, European Brown Hare Syndrome Virus (EBHSV) causes a disease lethal to brown hares Lepus europaeus. Associations of EBHSV infection, sex, age, body condition and spleen mass were studied in hares collected, from the prefectures of Chalkidiki and Thessaloniki, Hellas, during the hunting season. Data showed a sex-biased prevalence with twice as many males infected with EBHSV than females (P = 0.061), indicating a sexual dimorphism in disease exposure or susceptibility. EBHSV infection was not related to hare body condition (P = 0.853). Adults had significantly greater spleen mass than young hares (P = 0.003), indicating past infections. Significantly greater spleen mass was found in hares positive for EBHSV (P = 0.010) and a negative relation was found between spleen mass and body condition (P = 0.038), indicating energy consumption for immune defense investment. The present study is one of the few that examine the relevance of individual risk factors in understanding patterns of viral infections in natural populations of wild animals.

The effect of prolonged aflatoxin B 1 (AFB 1 ) administration on blood serum oestradiol-17β and progesterone concentrations in goats during the anoestrus period was investigated. Thirty Greek indigenous goats were used; 10 goats received, per os , 50 μg (treated group T50) and 10 goats received 100 &μg (treated group T100) AFB 1 /day/head, respectively, for 31 days, while 10 goats served as controls (C). Blood samples were collected from each goat twice a week to determine serum oestradiol-17β and progesterone concentrations by radioimmunoassay. Multiple linear regression analysis revealed a significant (P < 0.05) positive dependence of blood serum oestradiol-17β concentration over group (C = 0, T50 = 50, T100 = 100), in a dose-dependent manner, and a significant (P < 0.05) negative dependence over time (0-31 days, from the onset to the end of AFB 1 administration). Linear regression analysis revealed a significant (P < 0.05) positive dependence of blood serum progesterone concentration over group (C = 0, T50 = 50, T100 = 100), in a dose-dependent manner. Pearson's correlation coefficient revealed a significant (P < 0.05) positive correlation between blood serum oestradiol-17β and progesterone concentration, but only in the goats of the control group. In conclusion, the prolonged administration of 100 or even of 50 μg AFB 1 /day/head during the anoestrus period, increased blood serum oestradiol-17β and progesterone concentrations of goats, in a dose-dependent manner, and in the treated groups disturbed the positive correlation between oestradiol-17β and progesterone concentration shown in the goats of the control group.

The purpose of the present study was to investigate whether the electrical resistance values of the cer - vical mucus and/or blood οestradiol-17β and progesterone concentrations at mating, after oestrous synchronisation during the anoestrous or oestrous period in ewes, are related to fertility. Oestrus was induced by medroxyprogesterone acetate-impregnated intravaginal sponges (Veramix, Upjohn, USA), either for 12 days (12-day group; 24 Kymi and 28 Chios) or for 14 days (14-day group; 24 Kymi and 28 Chios), plus 400 IU equine chorionic gonadotropin (Intergonan, Intervet, the Netherlands) i.m., at the time of sponge removal. In each case, one blood sample was collected from all ewes at sponge insertion, at sponge removal and at oestrus, for the determination of serum οestradiol-17β and progesterone concentrations, using a radioimmunoassay. The electrical resistance of the cervical mucus was meas - ured once, just after oestrus detection by teaser rams, using a digital heat detector. All ewes found to be in oestrus were mated to fertile rams. Pregnancy was determined by transabdominal ultrasonography 35-40 days after mating. Kymi ewes were in anoestrous, while those of Chios were in the oestrous period. The 14-day treatment resulted in better outcome as concerns oestrus detection and pregnancy rate than the 12-day treatment, in either oestrous or anoestrous period ewes. Oestradiol-17β concentrations at sponge removal and at oestrus were lower ( P < 0.05) in the 14-day group than in the 12-day group, in both oestrous or anoestrous period ewes. Progesterone concentrations at sponge insertion and removal were higher ( P < 0.05), while progesterone concentrations and electrical resistance values of the cervical mucus at oestrus were lower ( P < 0.05) in ewes of both groups who conceived compared to those that did not, either in oestrous or in the anoestrous period. Linear regression analysis revealed a positive relation between the electrical resistance values of the cervical mucus and blood serum progesterone concentrations in both oestrous or anoestrous period ewes. The electrical resistance of cervical mucus could be useful for the detection of the proper time for mating after oestrous synchronisation in ewes.

Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue-type plasminogen activator (t-PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus-oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t-PA and/or its inhibitor epsilon-aminocaproic acid (control, t-PA, t-PA+ε-ACA, ε-ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t-PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε-ACA. PAA in the treated group was significantly reduced by ε-ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t-PA-modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t-PA. In conclusion, it appears that excessive t-PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena.

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.

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 (P0.05). 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.

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 (P0.05) and FT (P0.005) 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; P0.025). 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; P0.005) and FT (46.2%; 36/78 vs. 71.0%; 44/62; P0.01) spermatozoa. Both extenders were similarly effective in preserving pregnancy rates of mature ram semen (P0.05). 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 (P0.001). 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.

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.

Η πρόβλeψη της γονιμοποιητικής ικανότητας eνός κριού χρησιμοποιώντας in vitro δοκιμές, θα μπορούσe να έχeι μeγάλη σημασία για την αναπαραγωγική διαχeίριση σe ποίμνια προβάτων. Υπάρχουν πολλές διαδικασίeς in vitro που διατίθeνται για την αξιολόγηση της ποιότητας του σπέρματος και της γονιμοποιητικής ικανότητας, και η μέθοδος που θα eπιλeγeί eξαρτάται από το στόχο της αξιολόγησης του σπέρματος και των διαθέσιμων πόρων. Η αξιολόγηση της γονιμοποιητικής ικανότητας του σπέρματος in vitro, κατά αρχάς αναπτύχθηκe για την eφαρμογή της τeχνητής σπeρματέγχυσης (ΑΙ) και μeτέπeιτα για την eφαρμογή της τeχνικής της in vitro γονιμοποίησης (IVF). Οι τeχνικές της IVF eπιτρέπουν την eκτίμηση της γονιμοποιητικής ικανότητας του σπέρματος μe το να eκτιμούν την ικανότητα των σπeρματοζωαρίων να διeισδύουν και να γονιμοποιήσουν ώριμα ωάρια in vitro και μe το ποσοστό παραγωγής eμβρύων μeτά από in vitro γονιμοποίηση και καλλιέργeια των γονιμοποιημένων ωαρίων. Στην παρούσα βιβλιογραφική ανασκόπηση παρουσιάζονται συνοπτικά μeλέτeς που πραγματοποιήθηκαν in vitro, σe μια προσπάθeια για τη δημιουργία μιας eργαστηριακής δοκιμής που μe ακρίβeια να αξιολογeί, και ακόμη πeρισσότeρο να προβλέπeι την γονιμοποιητική ικανότητα σπέρματος κριών.

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.

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

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.

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.

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).