Evangelos Kiossis

Prenatal Estrogen and Progesterone Deprivation Impairs Alveolar Formation and Fluid Clearance in Newborn Piglets

Exposure to high levels of estradiol (E2) and progesterone (P) derived from the fetoplacentomaternal unit during the last trimester of pregnancy may play a crucial role in prenatal lung development and immediate postnatal alveolar fluid clearance (AFC). To measure prenatal alveolar formation and postnatal amiloride-sensitive AFC after pharmacological deprivation of E2 and P in utero, fetuses from five sows received an intramuscular depot injection of the E2 receptor blocker ICI 182.780 (ICI) and the P receptor blocker RTI 3021-022 (RTI) and fetuses of five other sows received a placebo injection (control group) during a laparotomy at 90 d of gestation (term gestation, 115 d). Piglets were delivered by cesarean section on d 114 of gestation. Of 95 live-born piglets, 35 were mechanically ventilated. The airways of the right lower lobe were isolated by a balloon catheter wedged in the bronchus and 5% albumin in 0.9% NaCl with or without 1 mmol/L amiloride was instilled. Amiloride-sensitive AFC was calculated from the protein concentration changes in fluid recovered after 120 min as the percentage of absorbed fluid. Lungs were removed under standardized conditions to perform alveolar counts. Prenatal treatment with ICI and RTI resulted in a significantly lower amiloride-sensitive AFC (median, 31%; min-max, –4–58) than placebo (74%, 18–231). Median alveolar counts per visual field were significantly lower in piglets that were exposed to ICI and RTI (38, 21–78) compared with placebo (56, 32–113). We conclude that prenatal E2 and P deprivation significantly impaired alveolar formation and amiloride-sensitive AFC.

Evidence of Schmallenberg virus circulation in ruminants in Greece.

During March 2013, we investigated the presence and the levels of Schmallenberg virus (SBV) circulation in three dairy cow herds and three sheep flocks in Central Macedonia, Greece. In two cow herds, a high number of abortions had been observed during the winter. Six bulk-tank milk samples and 147 individual sera were screened for SBV-specific antibodies by ELISA. Positive reactions were obtained from 5 out of 6 bulk-tank milk samples, 58 out of 90 sera from the 3 cow herds, and 2 sera from 2 of the 3 sheep flocks. Twenty-two ELISA-positive sera were tested by serum neutralization test (SNT). SNT confirmed the presence of neutralizing antibodies against SBV in all samples tested, with titers ranging between 1:32 and ≥1:256. No neutralizing antibodies against Akabane virus (AKAV) or Shamonda virus (SHAV) were detected, indicating that neutralizing antibodies against SBV do not cross react with AKAV or SHAV in SNT. ELISA testing of bulk-tank milk samples proved to be convenient and reliable. None of the tested sera was found positive for SBV by real-time RT-PCR, indicating that the sampling was conducted past the viremia stage. This is the first report of SBV circulation in Greece.

Chromatin integrity of ram spermatozoa. Relationships to annual fluctuations of scrotal surface temperature and temperature-humidity index

The objective of the present study was to explore the potential relationships of ovine sperm chromatin integrity, quantified using the sperm chromatin structure assay (SCSA), to the heat load of the scrotum and the discomfort felt by the animals because of fluctuations of microclimatic factors at different time periods before ejaculation. Ejaculates were collected once per week from five Chios rams and four East Friesian rams for 12 months and stored in liquid nitrogen. Frozen-thawed semen samples were analyzed using the SCSA, to determine the DNA fragmentation index (DFI) and the percentage of cells outside the main sperm population (%DFI) in each one of the samples. Scrotal surface temperature (SST) of each ram was measured using an infrared thermometer on a daily basis. Ambient air temperature and relative humidity were recorded at hourly intervals throughout the experimental period and temperature-humidity index (THI) was used to assess the discomfort felt by the rams. Mean values of SST (SST mean) and THI (THI mean) were computed for eight different time periods (up to 61 days) preceding each ejaculation day (Day 0). A linear mixed-effect model analysis was performed to describe the relation of SCSA parameters to collection month, SST mean, and THI mean of different time periods before ejaculation. The results of the statistical analysis revealed a relation of %DFI to the SST mean of the last 12 days preceding ejaculation, namely the period that resembled the phase of epididymal maturation. On the contrary, the variation of DFI was most adequately described by the linear mixed-effect model applied for Days 54 to 48 before ejaculation, which resembled the phase of spermatogonial mitoses. The effect of collection month was significant for DFI and %DFI, with semen samples collected in September and February exhibiting the lowest DFI values; a less profound seasonal pattern was detected for %DFI. The effect of THI mean on DFI and %DFI was proven nonsignificant in regard to all time periods. In conclusion, a relation of SCSA parameters to SST mean of different periods before ejaculation was shown in the present study, implying an effect of scrotal microenvironment on intratesticular and epididymal sperm population. In contrast, we failed to detect any effect of microclimate-induced discomfort felt by the animals on the chromatin integrity of frozen-thawed ram spermatozoa.

Effects of α- and β-adrenergic receptor stimulation and oxytocin receptor blockade on milking characteristics in dairy cows before and after removal of the teat sphincter

Alpha (alpha)- and beta (beta)-adrenergic receptors in the bovine mammary gland are mainly present in the teat muscles and in the region where large milk ducts reach the cisternal cavities. The aim of the study was to test the hypothesis that the region of the large mammary ducts is the most important location of alpha- and beta-adrenergic receptor stimulation affecting milk ejection and milk removal. Effects of alpha- and beta-adrenergic receptor stimulation and of oxytocin (OT) receptor blockade on milking characteristics were tested in six cows. Milk flow was measured before and after the distal part of one teat, including the teat canal and teat sphincter, had been partly amputated. Before the operation, milk yield and peak flow rate decreased during alpha-adrenergic receptor stimulation and during the OT receptor blockade, and increased during beta-adrenergic stimulation. After removal of the teat tip, relations of milk yield and peak flow rates after administration of alpha- and beta-agonists and after application of an OT receptor blocking agent were similar to those before operation. Only total milk yield had decreased in the teat-amputated quarter owing to unhindered flow of cisternal milk before cluster attachment. Since responses to alpha- and beta-adrenergic receptor stimulation as well as to OT receptor blockade do not differ with or without the teat sphincter, it is concluded that milk flow is mainly influenced by the muscle tone of the large mammary ducts.

Gender-related effects of prenatal administration of estrogen and progesterone receptor antagonists on VEGF and surfactant-proteins and on alveolarisation in the developing piglet lung.

BACKGROUND Vascular endothelial growth factor (VEGF) is essential for embryonic lung development and has been shown to be regulated by estradiol (E2) and progesterone (P). AIM To investigate the effects of prenatal E2 and P withdrawal by specific receptor antagonists on the mRNA expression of VEGF, surfactant proteins (SP-B and SP-C) and on alveolarisation in lung tissue of male and female pig fetuses. METHODS Fetuses from 10 sows were randomized to receive either both an intramuscular injection of the E2 receptor blocker ICI 182.780 and the P receptor blocker RTI 3021-022 (ICI+RTI, n=5) or a placebo injection (n=5) at 90 days of gestation (DOG, 115=term). After delivery by cesarean section on 114 DOG, tissue of the left lingula of the piglets lung (28 placebo, 26 ICI+RTI) was obtained to determine the mRNA expression of VEGF, SP-B and SP-C. Lungs from 15 placebo and 14 ICI+RTI group piglets were removed and alveolar counts performed. RESULTS The ICI+RTI group showed significantly lower SP-C mRNA expression and alveolar counts compared to the placebo group (p=0.04 and 0.03, respectively). Diminished alveolarisation in the ICI+RTI group was mainly due to the reduction of alveolar counts in male piglets (p=0.02). Within the placebo group VEGF and SP-B mRNA expression in male piglets were significantly lower compared to female piglets (p=0.01 and 0.004, respectively). ICI+RTI treatment abolished this gender-related difference. CONCLUSION Estradiol and P antagonism affected gender-related differences of key proteins for pulmonary function and development and especially in males was associated with diminished alveolarisation.

Lag effect of microclimatic conditions on DNA integrity of frozen–thawed bovine sperm

The objective of this study was to investigate the effect of microclimatic conditions on DNA integrity of bovine sperm. DNA fragmentation of frozen-thawed sperm was analyzed in monthly semen samples of nine AI bulls ejaculating in weekly routine for one year. The extent of DNA fragmentation was determined using the Sperm Chromatin Structure Assay (SCSA™) and quantified by the DNA fragmentation index (DFI) and the percentage of sperm showing high DFI values (%DFI) immediately (0h) and 3h (3h) after thawing. Microclimatic factors (ambient air temperature, relative humidity, dew point) were recorded in hourly intervals throughout the year. Their mean values were calculated for seven different time periods (up to day 56) preceding ejaculation (day 0). DFI (P<0.01) and its relative change between 0h and 3h (P<0.05) were related to the set of microclimatic parameters recorded the last 11 days and from day 49 to 43 prior to ejaculation, respectively. A significant relationship was detected between %DFI and microclimatic parameters of days 35-29 preceding ejaculation (P<0.05), while the degree of change of %DFI from 0h to 3h was related to microclimatic parameters recorded from day 28 to 22 before ejaculation (P<0.05). Dew point and relative humidity appeared to be the strongest and weakest predictors of DNA integrity, respectively. In conclusion, the results of the present study showed a lag effect of microclimate on DNA integrity of frozen-thawed bovine sperm. However, microclimatic parameters of a single time period before ejaculation could not be identified as the source of variation of different SCSA parameters.

Efficiency Assessment of a Swift Method to Enhance Substandard Viability Ram Ejaculates

Semen availability in ram semen processing facilities is of great importance for the genetic improvement of sheep. Accordingly, any method that would increase sperm viability in low viability ejaculates could be useful. In this study, the possibility of a glass beads filtration method by estimating the beads total surface provided (TSP) for adhesion of spermatozoa, was evaluated. Initially, two different TSP (102 and 154 cm(2)) achieved by various sizes of beads (1500, 2000 and 3000 microm) were tested and no significant difference in sperm viability improvement was noticed for the same TSP by different beads (p > 0.05). Next optimization tests were performed in which three different funnels were used for filtration at a standard TSP (154 cm(2)). The pear-shaped funnel was found to be the most appropriate for filtration, as semen volume recovery and sperm viability improvement were more pronounced (p < 0.05). Finally, filtration tests were conducted with pear-shaped funnels with different TSP (102 and 154 cm(2)) obtained by the aforementioned beads sizes (1500, 2000 and 3000 microm) in equal aliquots. Total surface provided of 102 cm(2) proved to be the more appropriate for filtration than 154 cm(2), as shown by the significant improvement of sperm viability (p < 0.01) and the significantly higher filtrate semen volume (p < 0.05). In conclusion, ram sperm viability improvement by more than 20% of its initial value and semen volume recovery by more than 60%, along with the fact that the total filtration time did not exceed 6 min in any case, suggest that through further development this method could be successfully used during ram semen processing.

Aetiology of reduced milk ejection in cows after transport and the use of a long-acting analogue of oxytocin for prophylaxis

Milk flow was recorded in 21 cows for three days after they were admitted to a large animal hospital. When the spontaneous flow of milk had stopped, a physiological dose (1 iu) of oxytocin was administered intravenously. Five of the cows were, in addition, treated with 0.35 mg of a long-acting analogue of oxytocin (carbetocin) one hour before the first milking after they were admitted. In the 16 cows not treated with carbetocin, only about 30 per cent of the total milk yield was released spontaneously on the first day, and the injection of 1 iu of oxytocin released approximately another 60 per cent of the total milk yield. On the second day, the proportion of the total milk yield released spontaneously increased and the fraction released after the injection of 1 iu oxytocin decreased. In contrast, the five cows treated with carbetocin released on average 94 per cent of the total milk yield spontaneously during the first milking.

Supplementation of lactating ewes with a glucogenic preparation or β-carotene in mid-to late lactation on subsequent milk yield and luteinizing hormone secretion

The effects of a glucogenic preparation (Nutrimix®) or β-carotene (Rovimix® beta carotene) supplementation of ewes during mid- to late-lactation on their milk yield were assessed in a study where 66 Chios-breed ewes were allocated into three equal (n = 22) groups. Ewes in group A received daily a glucogenic preparation and those in group B β-carotene, starting 35 d before estrus synchronization until 7 d after artificial insemination (supplementation period); ewes in group C were untreated controls. The estrus cycle was synchronized and double AI was carried out 48 and 60 h after withdrawal of progestagen sponges. Milk yield was measured daily during the supplementation period. Blood samples were collected at 2-h intervals, from 28 to 54 h after sponge withdrawal; the concentration of plasma luteinizing hormone (LH) was measured. Increased milk yield was recorded after supplementation with the glucogenic preparation (P 0.05). There were no differences in LH concentrat...

Combined and breed‐specific RIs for hematologic, biochemical, and hormonal analytes in Chios and Florina adult rams

BACKGROUND Clinical pathology investigations are essential to evaluate the health status and practice management of ruminants. Appropriate and accurate RIs are required for the interpretation of laboratory results. Despite substantial phenotypic differences and management-related disparities between male and female sheep, RIs in rams are sparse and limited by small reference populations and outdated methods. OBJECTIVES The purpose of this study was to establish RIs for hematologic, biochemical, and hormonal analytes in adult rams using controlled preanalytic and analytic procedures, and to investigate whether breed-dependent partitioning of RIs is required. METHODS Reference individuals were selected by a direct a priori method. Blood was collected from 128 healthy adult rams representing 2 Greek breeds (64 Chios and 64 Florina). Hematologic analysis was performed on the Advia 120, while differential WBC counts were manually performed on Giemsa-stained blood smears. Flexor E, AVL 9180 Electrolyte Analyzer, and Immulite 1000 were used for biochemical and hormonal analyses, respectively. Combined and breed-specific RIs were calculated using nonparametric and robust methods, respectively. RESULTS Five outliers were detected based on laboratory results. Partitioning of RIs, based on both statistical and nonstatistical criteria, was recommended for some of the analytes. CONCLUSIONS The RIs are in some agreement with those in the literature. The observed differences may be attributed to preanalytic and analytic factors. The RIs determined in this study can be used as a guide for the interpretation of laboratory results in rams and can potentially be adopted by veterinary laboratories, provided that similar equipment and reagents are used.