Experiences from the 2014 outbreak of bluetongue in Greece.

Objective of this paper was to review relevant work and to present a general account of the bluetongue outbreak, which occurred in Greece in 2014. In total, 2895 outbreaks of the disease have been reported by the veterinary authorities of Greece; sheep, goats and cattle were affected with officially reported morbidity rates of 11.0%, 2.0% and 3.5%, respectively. No vaccinations were allowed and conservative measures were implemented to attempt to limit the disease, which at the end had expanded throughout the country. In field investigations, a significantly higher bluetongue morbidity rate (27.5%) in sheep has been reported. During that work, clinical anaemia was encountered, which was characterised as macrocytic, hypochromic, regenerative and non-haemolytic. Other investigations, which are reviewed in this paper, have described an outbreak of Citrobacter freundii-associated enteritis in newborn kids, offspring of goats subclinically infected with Bluetongue virus, increased rate of early embryonic deaths, reduced conception rates, increased incidence risk of mastitis and reduced milk yield in herds of subclinically-infected cattle and detection of the virus from hunter-harvested tissue samples of roe-deer. In 2015, vaccines against the disease have been licenced; vaccinations started in May 2015. Then, in 2015, only one outbreak of the disease was confirmed, which could have been the result of a combination of reasons acting concurrently to prevent further cases.

Effects of addition of tissue-type plasminogen activator in in vitro fertilization medium on bovine embryo development and quality.

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.

Ghrelin accelerates in vitro maturation of bovine oocytes.

Ghrelin, apart from its metabolic role, is nowadays considered as a basic regulator of reproductive functions of mammals, acting at central and gonadal levels. Here, we investigated for possible direct actions of ghrelin on in vitro maturation of bovine oocytes and for its effects on blastocyst yield and quality. In experiment 1, cumulus oocyte complexes (COCs) were matured in the presence of four different concentrations of ghrelin (0, 200, 800 and 2000 pg/ml). In vitro fertilization and embryo culture were carried out in the absence of ghrelin, and blastocyst formation rates were examined on days 7, 8 and 9. In experiment 2, only the 800 pg/ml dose of ghrelin was used. Four groups of COCs were matured for 18 or 24 h (C18, Ghr18, C24 and Ghr24), and subsequently, they were examined for oocyte nuclear maturation and cumulus layer expansion; blastocysts were produced as in experiment 1. The relative mRNA abundance of various genes related to metabolism, oxidation, developmental competence and apoptosis was examined in snap-frozen cumulus cells, oocytes and day-7 blastocysts. In experiment 1, ghrelin significantly suppressed blastocyst formation rates. In experiment 2, more ghrelin-treated oocytes matured for 18 h reached MII compared with controls, while no difference was observed when maturation lasted for 24 h. At 18 and 24 h, the cumulus layer was more expanded in ghrelin-treated COCs than in the controls. The blastocyst formation rate was higher in Ghr18 (27.7 ± 2.4%) compared with Ghr24 (17.5 ± 2.4%). Differences were detected in various genes' expression, indicating that in the presence of ghrelin, incubation of COCs for 24 h caused over-maturation (induced ageing) of oocytes, but formed blastocysts had a higher hatching rate compared with the controls. We infer that ghrelin exerts a specific and direct role on the oocyte, accelerating its maturational process.

The effects of a glyphosate-based herbicide on the bovine gametes during an in vitro embryo production model.

Roundup® (R), while it is the most used herbicide globally, and its residues are ubiquitous in urban and suburban areas, its impact on vertebrates' safety remains highly debated. Here, in three in vitro experiments, we investigated the effects of a very low dose (1 ppm) of R on the fertilization capacity and embryo development in cattle. In the first experiment, frozen-thawed bull semen exposed to R for 1 h exhibited reduced motility parameters but unaffected fertilization ability. However, after in vitro fertilization, the rates of embryo formation were significantly lower compared to the untreated controls. In the second experiment, oocytes exposed to R during in vitro maturation showed reduced cleavage rates, and the embryo yield on days 7, 8, and 9 of embryo culture was significantly lower than that of the controls. In the third experiment, oocytes were matured in the presence of R and in a medium containing both R and Zinc, chosen to offer antioxidant protection to the oocytes. Day-7 blastocysts were analyzed for the expression of genes associated with oxidative stress, apoptosis, and epigenetic reprogramming. Exposure to R markedly suppressed embryo formation rates compared to the controls. The combination of R with Zinc restored the blastocyst yield, which on days 8 and 9 was comparable to that of the controls and higher than the groups exposed only to R on all days. The gene expression analysis revealed that R promotes oxidative stress development, triggers apoptosis, and induces epigenetic changes in developing embryos, while zinc presence alleviates these adverse effects of R. These findings imply that even at very low doses, R could be highly toxic, leading to functional abnormalities in both gametes, potentially affecting fertility in both genders.

Effects of guaiazulene on in vitro bovine embryo production and on mRNA transcripts related to embryo quality.

Reactive oxygen species (ROS) are between the major contributors for the reduced rate of in vitro bovine embryo production. It is believed that they can cause abnormal meiosis of oocytes, developmental arrest or cell death of embryos. Reports on the effectiveness of various antioxidants on embryo yield are rather conflicting mainly due to the nature and the concentration of the substances used. Here we report the effects of guaiazulene--an exogenous antioxidant, without known properties that could interfere with the biological process of IVF--on embryo development and on the quality of the produced blastocysts. Bovine cumulus oocyte complexes (COCs) were aspirated from abattoir ovaries and COCs were matured in TCM199 with FCS and EGF at 39 °C under an atmosphere of 5% CO(2) in air, with maximum humidity. After 24 h oocytes were inseminated with frozen/thawed semen and co-incubated for further 24 h. Zygotes were cultured in groups of 25 in 25 µl of SOF with 5% FCS at 39 °C under an atmosphere of 5% CO(2) , 5% O(2) in air with maximum humidity. In the first experiment the maturation medium was modified with addition of 0.1 mM of G (n = 497), or 0.01 mM of guaiazulene (n = 468), 0.05% DMSO--the guaiazulene diluent (Control(+), n = 467), and 459 oocytes were used as Control(-). In the second experiment, the culture medium was modified with the addition of 0.1 mM of guaiazulene (n = 344), 0.01 mM of guaiazulene (n = 345), 0.05% DMSO (Control(+), n = 347) and 355 were the Control(-). Blastocyst yield was recorded on days 6, 7, 8 and 9. Day 7 blastocysts from each experiment and group were snap frozen and stored for mRNA extraction. Quantification of transcripts for mRNA of genes related to metabolism (AKR1B1, PTGS2, GADPH, SLC2A5, G6PD); oxidation (GPX1); and implantation (PLAC8) was carried out by real time quantitative RT-PCR. Data for embryo development and on transcript abundance were analysed by χ(2) and anova respectively. In the first experiment no differences were found between groups in terms of cleavage rate (Control(-): 74.20%; Control(+): 74.58%; 0.01 mM: 71.61%; 0.1 mM: 71.63%) or day 9 blastocyst yield (Control(-): 28.26%; Control(+): 25.80%; 0.01 mM: 25.86%; 0.1 mM: 25.25%). In the second experiment, cleavage rate tended to be higher in 0.01 mM group than in Control(-) (77.87% vs 71.41% respectively, p = 0.07). No other differences were detected in cleavage rate (Control(+): 71.32%; 0.1mM: 72.75%) or in the overall blastocyst yield on day 9 (Control(-): 25.50%; Control(+): 26.71%; 0.01 mm: 29.58%; 0.1 mM: 25.75%). In both experiments the relative abundance of genes studied varied between groups but these differences were not statistically significant. Our results imply that oxidation has minimal effect on the in vitro embryo production. Guaiazulene, a compound possessing no biological properties other than those of a strong antioxidant, while it increased cleavage rate, it failed to improve either the blastocyst formation rate, or the quality of the produced embryos under 5% O(2) .

Clinical, ultrasonographic, bacteriological, cytological and histological findings during uterine involution in ewes with pregnancy toxaemia and subsequent reproductive efficiency.

Objectives were to evaluate characteristics of uterine involution in ewes with pregnancy toxaemia during gestation and to study effects on subsequent reproductive performance. Pregnancy toxaemia was induced in ewes (A) by feeding an energy-deficient diet as confirmed by detecting ß-hydroxybutyrate concentrations in blood indicative of this disorder. There was also a control group (C). Animals were evaluated until the 60th day post-partum using clinical and ultrasonographic examinations. Vaginal swab samples and uterine biopsy tissue samples were collected for bacteriological and cytological examination; biopsy samples were prepared for histological examination. Ewes were subsequently placed with rams and reproductive performance was ascertained. Post-partum, during the ultrasonographic examination of the uterus, ewes of Group A had caruncle and uterine lumen diameters, as well as a uterine thickness greater than ewes of Group C. Post-partum uterine blood flow volume was greater in ewes of the A than C group. Neutrophils predominated in vaginal samples, with the neutrophil proportion being less in ewes of Group A than C. There were no differences in the uterine involution process between groups. During the subsequent reproductive season, all the ewes of Group A lambed normally and produced viable lambs. It is concluded that there were no adverse effects on subsequent reproductive performance of ewes previously affected with pregnancy toxaemia, when appropriate health management was performed.

Oestradiol concentration as a predictor of ovarian response in FSH stimulated ewe-lambs.

We investigated the prediction of ovarian response using oestradiol determination, in 37 gonadotrophin stimulated Karagouniko ewe-lambs. Ovarian stimulation was induced by serial FSH administrations, and laparoscopic follicular aspiration (OPU) was conducted 12h after the last FSH injection. Oestradiol concentration was assessed in six blood samples collected prior to each FSH injection and in one sample collected prior to follicular aspiration. According to ovarian response, ewe-lambs were allotted in three groups: good, L1 (n=17); moderate, L2 (n=10); and poor, L3 (n=10). Based on the data obtained from 28 (75%) randomly selected animals, a statistical model was designed and tested on the remaining nine lambs for its ability to predict the probability of good ovarian response. From the 2nd sample, oestradiol concentration was constantly higher in group L1 in comparison with L3 lambs (all p-values for the contrasts were

The association of sub-clinical paratuberculosis with the fertility of Greek dairy ewes and goats varies with parity.

Our cross-sectional study investigated the association of sub-clinical Mycobacterium avium subsp. paratuberculosis (MAP) infection with failing to produce a live offspring the season of lambing/kidding (November 2001 to January 2002) before testing (in April-May 2002), in four dairy-sheep and/or goat flocks in Greece (369 animals >or=1.5-year-old). From each selected animal 10 ml of blood and 10 g of feces from the rectum were obtained. The harvested sera were tested for antibodies to MAP with a commercial ELISA test kit; the feces were cultured on Herrold's egg-yolk medium supplemented with mycobactin J and antibiotics. An animal was considered sub-clinically infected when found either seropositive or culture positive. The true prevalence of sub-clinically infected animals, adjusted for the sensitivity and specificity of the parallel testing, was 14% (0.1-28%) and 35.9% (9.2-62.7%) in sheep and goats, respectively. The association of fertility of sheep and goats with sub-clinical paratuberculosis was investigated in random-effects logistic models. Sub-clinically infected animals (compared to uninfected) had OR for live offspring the previous year of 5.4 for parity <4, OR=0.05 for parity >6, and a non-significant OR for the middle parity category.

Oocyte pick-up in juvenile lambs affects neither onset of puberty nor their future fertility.

A study was designed to evaluate whether the time of onset of puberty and fertility of young ewe lambs would be affected by oocyte pick-up conducted in single or repeated sessions during the first months of lambs' live. Five groups of lambs from the Karagouniko breed were used (A-E each n=12). In group A no treatments were applied (control group) while, laparoscopical follicular aspiration (OPU) was performed early in the third, fourth and fifth month of lambs age (groups C-E, respectively). From the second to fifth month of their age, group B lambs were aspirated four times in monthly intervals. All lambs were weighed at birth, weaning, at second month and monthly thereafter until the eighth month of age. Progesterone priming and ovarian stimulation by serial FSH administrations proceeded each OPU session. To determine onset of puberty blood progesterone concentration was assayed in samples collected initially every week and after the seventh month of age twice weekly. From the seventh month a fertile ram was introduced in each group and oestrous behavior/mating was daily monitored and recorded. Pregnancy diagnosis was carried out by transabdominal ultrasound scanning 55 days after rams' removal. At the fourth and fifth month of age group B lambs were lighter (p<0.05) than controls, but this difference was later equalized. The time of onset of puberty did not differ between groups (p=0.069) and ranged between 224 and 270 days. Some animals (n=15) entered puberty with a full-length luteal phase having progesterone concentration greater than 1ng/ml, while others (n=32) exhibited one or two short luteal phases before luteal length restoration. During the first breeding season 41 animals were fertilized and maintained pregnancy to term, without noticeable differences between groups (p=0.555). During the second breeding season, all ewes were naturally served and lambed at the expected time. It is concluded that OPU in young dairy lambs does not affect the time of onset of puberty, the endocrine profile of the lambs and it does not compromise their future fertility even if it is applied at four successive months.

The activity of three glycosidases (ß-Ν-acetyloglucosaminidase, α-mannosidase, and ß-galactosidase) in the follicular fluid and in the maturation medium affects bovine oocyte maturation.

We studied the role of follicular fluid's (FF) glycosidase (α-mannosidase [α-ΜΑΝ], ß-Ν-acetyloglucosaminidase [NAGASE], ß-galactosidase [ß-GAL]) activity during IVM of bovine oocytes. Oocytes were allocated into two groups according to the follicular size (small follicle [SF]: 2-5 mm, large follicle [LF]: >5-8 mm). In experiment 1, cumulus-oocyte complexes (COCs) quality was evaluated according to morphologic criteria (grades A, B-C, D); oocyte (n = 801) nuclear maturation was assessed after 24 hours of incubation. Bovine embryos were produced in vitro in groups (experiment 2, n = 1503 oocytes) or individually (experiment 3, n = 50 oocytes). More grade-A and -BC COCs were collected from SF and LF groups, respectively (P < 0.05). Maturation rate (experiment 1) and cleavage rate (experiments 2 and 3) were similar in SF and LF groups. Activity of all glycosidases in FF was higher (P < 0.05) in SF group than in LF group, whereas in maturation medium of SF group it was, overall, significantly lower than in that of LF (experiments 2 and 3). In FF of SF group, NAGASE positively associated with grade-A oocytes and negatively with BC oocytes; increased ß-GAL was associated with degenerated oocytes. Cleavage rate in LF group, related negatively to NAGASE and positively to α-MAN in maturation medium. These results indicate that during maturation, COCs release NAGASE and consume ß-GAL, but differences probably exist between individual and group maturation.

Ghrelin suppresses the GnRH-induced preovulatory gonadotropin surge in dairy heifers.

Ghrelin, a known growth hormone (GH) secretagogue, alters gonadotropin secretion in many species. Our objectives were to study the effects of ghrelin, on GH, LH, FSH secretion, and on luteal function of the ensuing estrous cycle in cattle. The estrous cycles of eight heifers were synchronized with progesteron releasing intravaginal device, and ovulation was induced with GnRH. Eight animals were treated with 1.5 µg kg(-1) bovine ghrelin (group Ghr, n = 4) or saline (group C, n = 4). Starting with the first ghrelin injection, 13 blood samples were collected over a 4-hour period for the determination of ghrelin, GH, LH, and FSH concentration. Progesterone levels were measured in samples collected every other day after estrus expression. Data were analyzed by repeated measures of ANOVA followed by Bonferroni post hoc testing and t test. In group Ghr, ghrelin concentration increased significantly 15 minutes after the first injection and remained in elevated levels until the 90th minute after the last injection. At the time of third ghrelin injection, GH was significantly higher in the Ghr group compared with C (17.1 ± 1.3 vs. 2.6 ± 0.3 ng mL(-1), P < 0.0001). Similar differences were found for the next three samples collected 15, 30, and 60 minutes later; no difference was evident after 90 minutes. In group Ghr, the area under the curve for LH and FSH were significantly reduced compared with the ones of group C (266 ± 10.3 vs. 331.9 ± 7.3, P = 0.007 and 102.3 ± 2.0 vs. 134.9 ± 5.5, P < 0.005 for LH and FSH respectively). At particular time points the concentration of the two gonadotrophins in group Ghr was significantly lower than those of group C (15, 30, 45, 75, and 90 and 60, 75, 90, 120, and 150 minutes after GnRH administration for LH and FSH respectively). The duration of the following estrous cycle was shorter (P = 0.004) in group Ghr (19.0 ± 0.4 days) compared with C (21.8 ± 0.5 days). In days 4, 6, 8, 10, and 14, progesterone concentration was lower (P < 0.05) in group Ghr compared with C; similarly the progesterone area under the curve for group Ghr (113.1 ± 4.8) was suppressed (P = 0.007) compared with that of C (141 ± 4.8). These results imply that ghrelin acts on pituitary causing impaired response to the GnRH stimulus, and it is likely to affect luteinization of the cellular compartment of the preovulatory follicle, and/or to suppress steroidogenetic activity of the luteal cells.

Luteal stage dependence of pituitary response to gonadotrophin-releasing hormone in cyclic dairy ewes subjected to synchronisation of ovulation.

Possible hormonal aberrations precluding conception or maintenance of pregnancy in dairy ewes subjected to ovulation synchronisation were investigated in this study. The pituitary response to exogenous gonadotrophin-releasing hormone (GnRH) was tested at different luteal stages in 36 ewes. Oestruses were synchronised by using progestagen-impregnated sponges and the animals were randomly allotted into one of three treatment groups (A, B and C; n = 12 for each). Treatments commenced on Days 4, 9 and 14 of the new cycle (oestrus was defined as Day 0). Ewes were given two GnRH injections, 5 days before and 36 h after a prostaglandin F2+/- (PGF2+/-) injection, and the animals were inseminated 12-14 h after the second GnRH injection (modified OVSYNCH). For luteinising hormone (LH) determination blood samples were withdrawn from six ewes of each group at the time of GnRH administration, and 30, 90, 180, 270 and 360 min later. Progesterone was assayed in samples taken every other day starting from oestrus and for 17 days after the second GnRH injection, and in an additional sample collected on the day of insemination. After the first GnRH injection, the LH concentration was higher in Group C than in Groups B and A (mean +/- s.d.: 64.8 +/- 10.0 ng mL(-1), 41.3 +/- 3.7 ng mL(-1) and 24.6 +/- 9.0 ng mL(-1), respectively; P < 0.05), whereas after the second GnRH injection a uniform LH release was found in all groups. PGF2+/- caused a significant decrease in progesterone (P4) concentration in all groups; however, at artificial insemination ewes that conceived had significantly lower P4 concentration in comparison with those that failed to conceive. As early as Day 5, pregnant animals had higher P4 concentrations than non-pregnant animals. Overall, 21 animals conceived (seven, nine and five ewes from Groups A, B and C, respectively). These results indicate that the proposed protocol is equally effective in inducing a preovulatory LH surge at any stage of the luteal phase, and that elevated P4 concentration along with a delayed P4 increase should be considered as a causative factor for inability to conceive.

Prevalence of BVDV infection in Greek dairy herds.

Thirty-nine Greek dairy herds, totalling 6333 cattle, enrolled in a voluntary bovine viral diarrhoea virus (BVDV) eradication programme based on the identification and removal of persistently infected (PI) animals. The aim of this study was to estimate the prevalences of BVD antigen-positive and PI animals, and investigate the significance of the associations between the prevalence estimates and herd size. Initially, all animals were bled and examined for BVDV, using an antigen ELISA. A second sample was collected from the positive animals, after a period of at least three weeks. Animals retested positive were classified as PI. Antigen positive and PI animals were detected in all herds. The respective mean prevalences, adjusted for the test's accuracy and the herd-clustering effect, were 14% (95%CI: 11-18%) and 1.3% (0.8-1.8%), respectively. Herd size was not associated with the prevalence of antigen-positive or PI animals.

Incidence risk and clinical features of retention of foetal membranes in ewes in 28 flocks in southern Greece.

In a field investigation of 28 flocks in southern Greece, 7660 lambings were monitored. Retention of foetal membranes (defined as failure to expel the foetal membranes within 6 h of lambing the last lamb) was recorded in 92 ewes. The median within-flock incidence risk was 1.25% (range: 0-1.9%). The heterogeneity of the risk among flocks was not significant (p = 0.99); no correlation was found between the incidence risk and the flock size (r(sp) = 0.33, p = 0.09). Overall, the median duration of retention was 72 h (range: 9-288 h); it did not differ among flocks (p = 0.89) and was not correlated with flock size (r(sp) = 0.24, p = 0.27). During the initial stage of retention the membranes were fleshy and humid; the cotyledons were thick and congested. Progressively, the membranes became thin, dry and stringy; the cotyledons shrunk and were pale. Finally the membranes dropped as a mass. In ewes with retention for >12 h, a variety of accompanying signs was recorded: straining (in 22% of the ewes with retention), vulval oedema and reddening (in 16%), anorexia (in 13%), recumbency (in 13%) and increased temperature (in 12%) were the most frequent ones. Overall, the median clinical score of the disorder was '2' (range: '1'-'5'); it did not differ among flocks (p = 0.98) and was not correlated with flock size (r(sp) = 0.26, p = 0.25). In 4% of the ewes with retention for <4 days accompanying signs were recorded (median clinical score: '2'), whilst in 80% of the ewes with retention for > or =4 days accompanying signs were recorded (median clinical score: '3'). This difference in the prevalence of clinical signs was significant (p < 0.0001).

A matched case-control study of factors associated with retention of fetal membranes in dairy ewes in Southern Greece.

We defined retention of fetal membranes (RFM) in dairy ewes as failure to expel the placenta within 6h after lambing the last lamb and designed a matched case-control study to identify factors that affect the risk of retention. For each ewe with RFM, the next ewe in the flock that lambed and expelled the placenta in <6h after lambing the last lamb was selected as control. Data analyzed included 92 pairs of ewes from 25 flocks comprising a total of 7275 ewes (median flocksize 270 ewes). Factors investigated for associations with RFM were induction of lambing, obstetrical assistance because of dystocia, parity, the number of liveborn lambs, the occurrence of stillbirth(s), the mean weight of the litter on the third day post-lambing and the occurrence of neonatal death in the litter. Conditional logistic regression indicated (1) that the risk of RFM increased linearly with increasing number of liveborn lambs and (2) that the risk of retention was 4-fold higher in ewes that received assistance at lambing than those that lambed normally.

Use of the polymerase chain reaction to sex the bovine fetus using cells recovered by ultrasound-guided fetal fluid aspiration.

The aim of this study was to carry out first trimester fetal sex diagnosis using the polymerase chain reaction (PCR) to amplify DNA from bovine fetal cells recovered by transvaginal ultrasound-guided uterine puncture and fetal fluid aspiration. For sex determination, a nested, allele-specific, PCR amplification of the bovine zfx and zfy gene fragments was utilised. The PCR assay was validated using fetal fluids recovered from uteri post mortem. Cells were harvested from the fetal fluids, genomic DNA extracted and the PCR assay applied. A technique which was developed for transvaginal ultrasound-guided follicle aspiration was modified to recover fetal fluid from live animals. Small volumes of fetal fluid (0.5-5 ml) were recovered between days 61-97 of gestation and the PCR assay applied. The gender determined by PCR of fetal fluid cells was in all cases confirmed by visual inspection (n = 15 abattoir specimens) or ultrasound scanning (n = 7 live animals). Fetal death, attributed to the introduction of intrauterine infection, occurred in 4/4 cows in the first series of aspirations but in only 1/3 heifers in the second series of aspirations.

Bacteriological and epidemiological findings during examination of the uterine content of ewes with retention of fetal membranes.

We included 92 pairs of ewes with or without retention of fetal membranes in a cohort study of 25 flocks in Southern Greece. We obtained two uterine content samples under aseptic conditions, by introducing a swab into the uterus of these ewes, on the 2nd-4th and the 5th-9th day after lambing. We used conventional bacteriological techniques to isolate and identify bacteria and to carry out antimicrobial agents susceptibility testing. The prevalence of bacterial intrauterine contamination among ewes with retention was 24% on the first and 46% on the second sampling (P < 0.0001) and that among ewes without retention was 8 and 2% (P > 0.05), respectively. Clinical signs accompanying the retention of fetal membranes were more frequently observed among ewes with intrauterine contamination than among those without (P = 0.0007). The odds of an ewe having an intrauterine contamination increased multiplicatively by 1.06 when the median duration of retention in the flock increased by 6 h. The principal bacteria isolated from the ewes with retention were Arcanobacterium pyogenes and Escherichia coli; 21% of 73 isolates tested were found resistant to at least one antimicrobial agent.

Fertilization and embryo recovery rates in superovulated chios ewes after laparoscopic intrauterine insemination.

Forty superovulated dairy ewes of the Greek Chios breed were used in an experiment to evaluate the efficiency of laparoscopic intrauterine insemination on fertilization and embryo recovery rates as well as embryo quality. Estrus was synchronized by intravaginal progestagen impregnated sponges and superovulation was induced by administration of 8.8 mg o-FSH i.m. following a standard 8 dose protocol. A small volume (0.3 mL) of diluted fresh ram semen was deposited in each uterine horn 24 to 28 h after onset of the estrus by a laparoscopic technique. The animals were allocated randomly into two groups (Group A and B) of 20 animals each. In Group A, embryos were recovered 18 to 24 h after the intrauterine insemination and in Group B on Day 6. The average number of corpora lutea was 12.8 +/- 1.2 and 11.5 +/- 1.1 (+/- SEM); the overall embryo recovery was 66.4% and 57% and the percentage of recovered fertilized ova was 81% and 82.8% in Groups A and B, respectively. More fertilized ova were collected per ewe from Group A (P < or = 0.1). Results indicated that in Chios breed, superovulation using homologous FSH combined with laparoscopic AI leads to good ovarian response with satisfactory results in fertilization, embryo recovery and quality of embryos. This could lead to improved and more efficient methods for obtaining large numbers of high quality oocytes and embryos for embryo transfer programs which could contribute to genetic improvement and increase of the population size.

Progesterone concentration as an indicator of ovarian response to superovulation in Chios ewes.

We investigated the prediction of the ovarian response to superovulation using progesterone (P4) determination in Chios ewes. During the estrus period. estrus synchronization and multiple ovulations were induced in 100 non-pregnant, non-lactating Chios ewes by a combination of FGA-impregnated intravaginal sponges and 8.8 mg of ovine FSH. Laparoscopic insemination was conducted 24-28 h after the onset of estrus. A concentration of P4 was determined on Day 5 of the estrous cycle and on Day 6 the ovarian response was evaluated by counting the corpus lutea (CL); subsequently, embryo collection was performed. According to the response of their ovaries, ewes were allocated into four groups: A (n = 30); B (n = 37); C (n = 22); D (n = 11), with minimal (0-3 CL), moderate (4-8 CL), good (9-13 CL) or extreme (> 13 CL) ovarian response, respectively. In groups C and D, the mean blood serum P4 concentration (23.2 and 27.3 ng/ml, respectively) was higher (P < 0.001) than that in groups A and B (4.6 and 13.1 ng/ml, respectively); no difference was detected in blood P4 concentration between groups C and D. A strong linear relation (F < 0.00005) was found between blood P4 concentration and the number of CL, as well as between blood P4 and a dummy variable corresponding to poor (< 4 CL) or moderate/good/extreme ovarian response (>3 CL). Our results indicate that based on blood P4 measurement, it is feasible to identify ewes that should show the highest embryo recovery, while it is impossible to predict the exact number of CL formed.

Ultrasonographic appearance of clinically healthy testicles and epididymides of rams.

The clinically healthy testicles and epididymides of 31 rams were imaged inside and outside the breeding period, by using a real time ultrasound scanner. A scanning technique based on multiple imaging planes from the caudal and the lateral surface of the genitalia was employed. Optimum imaging was achieved by using a 6.0 MHz frequency sector transducer. The testicular parenchyma appeared homogeneous with a coarse medium echo-pattern. The mediastinum testis was present in 87% of rams and 77% of testicles; its median echogenicity score was 2 (range: 0-3) among rams aged 13 months or older and 1 among rams aged less than 13 months (P = 0.001). The tail of the epididymis was always clearly visible; it appeared less echoic than the testicular parenchyma and with a heterogeneous structure. The epididymal body was not visible, whilst the epididymal head was consistently partially imaged. The pampiniform plexus was clearly imaged as a dome-shaped structure masking the upper part of the head of the epididymis. The scrotal septum was seen in lateral sonograms as a highly echogenic line between the testicles. The scrotal skin formed a thick hyper-echoic peripheral structure.