Synergistic Effect of Porcine Follicular Fluid and Dibutyryl Cyclic Adenosine Monophosphate on Development of Parthenogenetically Activated Oocytes from Pre‐Pubertal Gilts

This study investigated the effect of porcine follicular fluid (PFF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) during in vitro maturation (IVM) of porcine oocytes on meiotic maturation, fertilization and embryo development, and compared the effect of supplementing the embryo culture media with PFF or foetal bovine serum (FBS) on embryo development. Oocytes from pre‐pubertal gilts were IVM for 44 h, and parthenogenetically activated or in vitro‐fertilized. Embryos were cultured in porcine zygote medium (PZM3) for 7 days. Cleavage and blastocyst rates were evaluated at 48 h and 7 days of culture. The supplementation of the IVM medium with 25% PFF and 1 mm dbcAMP for the first 22 h resulted in more (p < 0.05) embryos developing to the blastocyst stage as compared with the inclusion of dbcAMP alone. The dbcAMP + PFF combination increased (p < 0.05) the average number of nuclei per blastocyst as compared with either of these components alone or in its absence. A synergistic effect of dbcAMP + PFF during IVM was also reflected in the capacity of oocytes to regulate sperm penetration and prevent polyspermy, as twice as many oocytes from the control group were penetrated by more than one sperm as compared with those matured in the presence of both dbcAMP and PFF. The supplementation of PZM3 with 10% FBS from days 5 to 7 of culture significantly improved the total cell quantity in embryos derived either from control or dbcAMP + PFF matured oocytes. There was no effect on the total cell quantity when FBS was replaced by the same concentration of PFF. These studies showed that dbcAMP, PFF and FBS can improve both the quantity (57.3% vs 41.5%) and quality (74.8 vs 33.3 nuclei) of porcine blastocysts derived from oocytes recovered of pre‐pubertal gilts.     

Diagnostic Accuracy of D‐Dimer and Other Peritoneal Fluid Analysis Measurements in Dairy Cows with Peritonitis

Background: Peritoneal fluid analysis in cattle traditionally includes the classic parameters despite the fact that they have only moderate diagnostic accuracy and often fail to identify the pathogenesis or etiological factors. Therefore additional parameters recently have been established to improve diagnostic precision. In a recent study, reference ranges for several of these parameters have been proposed in dairy cows. Hypothesis/Objectives: The aim of this observational study was to assess the diagnostic value of D‐Dimer and other measurements of peritoneal fluid analysis in dairy cows with peritonitis. Animals: The study included 110 Holstein‐Friesian cows grouped into cows with peritonitis (n = 47) and cows without peritonitis (n = 63). Methods: Peritoneal fluid was obtained by abdominocentesis. Total protein, albumin, glucose, cholesterol, fibrinogen, l ‐lactate, D‐Dimer, lactate dehydrogenase (LDH), alkaline phosphatase, creatine phosphokinase, white blood cell, and red blood cell were determined in peritoneal fluid and venous blood. Serum‐ascites albumin gradient (SAAG) and ratios of peritoneal fluid‐venous blood were calculated. Sensitivity (SN) and specificity (SP) were calculated and receiver operating characteristic curve analysis performed. Results: Peritoneal fluid D‐Dimer was most accurate in diagnosing peritonitis in cows (SN and SP>95.0%). Total protein concentration, LDH and LDH ratio, and SAAG had sensitivities between 49.0 and 67.1%, and specificities between 88.4 and 95.5%. A low‐peritoneal fluid glucose concentration was found to be highly indicative of septic peritonitis. Conclusions and Clinical Importance: Measurement of the recently introduced parameters may increase the diagnostic value of peritoneal fluid analysis and provide additional specific information. Therefore these measurements should be included in the routine procedure.     

Expression of Mitochondria‐Associated Genes (PPARGC1A,NRF‐1, BCL‐2 and BAX) in Follicular Development and Atresia of Goat Ovaries

Most follicles undergo atresia during the developmental process. Follicular atresia is predominantly regulated by apoptosis of granulosa cells, but the mechanism underlying apoptosis via the mitochondria‐dependent apoptotic pathway is unclear. We aimed to investigate whether the mitochondria‐associated genes peroxisome proliferator‐activated receptor‐gamma, coactivator1‐alpha (PPARGC1A), nuclear respiratory factor‐1 (NRF‐1), B‐cell CLL/lymphoma 2 (BCL‐2) and BCL2‐associated X protein (BAX) played a role in follicular atresia through this pathway. The four mitochondria‐associated proteins (PGC‐1α, which are encoded by the PPARGC1A gene, NRF‐1, BCL‐2 and BAX) mainly expressed in granulosa cells. The mRNA and protein levels of PPARGC1A/PGC‐1α and NRF‐1 in granulosa cells increased with the follicular development. These results showed that these genes may play a role in the regulation of the follicular development. In addition, compared with healthy follicles, the granulosa cell in atretic follicles had a reduced expression of NRF‐1, increased BAX expression and increased ratio of BAX to BCL‐2 expression. These results suggested that changes of the mitochondria‐associated gene expression patterns in granulosa cells may lead to follicular atresia during goat follicle development.     

First Evidence of Porcine Circovirus Type 2 (PCV‐2) Infection of Pigs in the Czech Republic by Semi‐Nested PCR

Three oligonucleotide primers for semi‐nested polymerase chain reaction (PCR) were designed according to already published sequences of porcine circovirus types 1 (PCV‐1) and 2 (PCV‐2) isolates. These primers were used to detect PCV‐2 DNA. A positive amplification reaction was visualized from a DNA suspension containing as few as 10 copies of virus DNA. In total, 77 samples of inguinal lymph nodes and nasal swabs from pigs in the Czech Republic were used to detect the virus. Thirty‐seven of them were positive for PCV‐2 DNA. In order to confirm specificity of the PCR reaction, seven DNA fragments were sequenced. Czech PCV sequences were found to have a 92–97% homology with other known PCV‐2 strains and only 80–83% homology with PCV‐1 strains.     

Optimizing Electrical Activation of Porcine Oocytes by Adjusting Pre‐ and Post‐Activation Mannitol Exposure Times

Modifying electrical activation conditions have been used to improve in vitro embryo production and development in pigs. However, there is insufficient information about correlations of porcine embryo development with oocyte pre‐ and post‐activation conditions. The purpose of this study was to compare the developmental rates of porcine oocytes subjected to different mannitol exposure times, either pre‐ or post‐electrical activation, and to elucidate the reason for the optimal mannitol exposure time. Mannitol exposure times around activation were adjusted as 0, 1, 2 or 3 min. Blastocyst development were checked on day 7. Exposure of oocytes to mannitol for 1 or 2 min before electrical activation produced significantly higher blastocyst rates than exposure for 0 or 3 min. There was no significant difference in blastocyst rates when activated oocytes were exposed to mannitol for 0, 1, 2 or 3 min after electrical activation. While exposure of oocytes to mannitol for 1 min pre‐ and 3 min post‐activation showed significantly higher blastocyst development than 0 min pre‐ and 0 min post‐activation. It also showed higher maintenance of normal oocyte morphology than exposure for 0 min pre‐ and 0 min post‐activation. In conclusion, exposure of oocytes to mannitol for 1 min pre‐ and 3 min post‐activation seems to be optimal for producing higher in vitro blastocyst development of porcine parthenogenetic embryos. The higher blastocyst development is correlated with higher maintenance of normal morphology in oocytes exposed to mannitol for 1 min pre‐ and 3 min post‐activation.     

Vitamin A and β-Carotene Levels in Plasma, Corpus Luteum and Follicular Fluid of Cyclic and Pregnant Cattle

This study was carried out to examine the relationship between the corpus luteum (CL) weight, CL and follicle diameters and progesterone, β‐carotene and vitamin A levels in reproductive organs of cattle obtained from the slaughterhouse. The β‐carotene and vitamin A levels were determined in plasma, CL and follicular fluid (FF) using a spectrophotometric method at different stages of the oestrous cycle (n=40) and at 3–6 months of pregnancy (n=10). The diameters of the CL and follicle were measured using ultrasonography. Plasma progesterone concentrations were determined by an enzyme immunoassay method. The vitamin A levels of the plasma, CL and FF were not related to each other. The highest plasma vitamin A levels were observed in the proestrus and oestrus, at which periods follicular activity dominates. The vitamin A levels in the CL and FF were negatively related to the weight and diameter of the CL and the diameter of follicle, respectively. In contrast to vitamin A, β‐carotene concentrations of plasma, CL and FF were significantly correlated with each other. The highest β‐carotene levels in the plasma, CL and FF were found during pregnancy when there is maximal luteal function, and the β‐carotene level of the CL was significantly correlated with the weight and diameter of CL. Furthermore, the intrafollicular β‐carotene level was negatively correlated with the follicle diameter. There was a positive correlation between plasma progesterone level and the weight and diameter of the CL, but a negative correlation between plasma progesterone level and follicle diameter. Moreover, plasma, FF and CL β‐carotene levels were positively correlated with plasma progesterone levels. This study revealed that β‐carotene levels in the plasma, CL and FF were influenced by the stage of the oestrous cycle or the pregnancy and were related to bovine luteal function without depending on vitamin A.     

Detection of Porcine Circovirus Type 2, Porcine Parvovirus and Porcine Pseudorabies Virus from Pigs with Postweaning Multisystemic Wasting Syndrome by Multiplex PCR

Multiplex PCR was established to detect porcine circovirus type 2 (PCV-2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV) and applied to samples from 137 piglets exhibiting clinical signs of postweaning multisystemic wasting syndrome (PMWS). PCV-2 DNA was detected from all samples. Moreover, 43 samples were positive for PPV but negative for PRV; 11 samples were positive for PRV but negative for PPV; and 35 samples were positive both for PPV and PRV. These results suggests that PCV-2 co-infection with PRV and PPV may play an important role in PMWS. Also, multiplex PCR is an appropriate candidate method for diagnosis of PCV-2, PRV and PPV simultaneously in field cases.     

Paraoxonase (PON) 1, 2 and 3 Expression in Granulosa Cells and PON1 Activity in Follicular Fluid of Dairy Cows

Normal metabolic activity in ovarian follicles may result in oxidative stress and damage to oocytes. The aim of this study was to evaluate expression of the natural anti‐oxidants paraoxonase (PON) 1, 2 and 3 in granulosa cells and PON1 activity in follicular fluid (FF) and plasma of dairy cows. For the first experiment, ovaries were collected from cows at slaughter, after which follicles were dissected and classified as oestrogen active (EAF) or atretic (ATF). Expression of PON1, PON2 and PON3 mRNA was evaluated in granulosa cells, and activity of PON1 was measured in FF. PON1 mRNA was undetectable in granulosa cells, PON2 mRNA expression was not different between follicle types, and PON3 mRNA tended to be higher in EAF (p = 0.11). The activity of PON1 in FF was higher (p = 0.01) for EAF (82.6 ± 8.0 kU/L) than ATF (53.9 ± 6.8 kU/L), as were high‐density lipoproteins (HDL), low‐density lipoproteins (LDL) and total cholesterol concentrations. In the second experiment, we aimed to compare plasma and FF PON1 activity in early lactation Holstein cows (n = 15) with pre‐ovulatory EAF. Activity of PON1 was twofold higher (p < 0.0001) in plasma (122.5 ± 11.1 kU/L) than in FF (61.4 ± 5.2 kU/L). Plasma concentrations were also higher (p < 0.0001) for HDL, LDL and total cholesterol when compared to FF. In conclusion, FF concentrations of PON1, HDL, LDL and total cholesterol were higher in healthy oestrogen active bovine follicles than in atretic follicles. PON1 was not expressed by granulosa cells indicating that high PON1 activity in bovine FF is apparently derived by transfer from blood in association with HDL.     

Altered Expression of 3β‐HSD,CYP17 and 17β‐HSD in the Foetal Porcine Gonads in Response to Anti‐androgen Flutamide Exposure

We investigated whether the limited access to androgens during late prenatal period alters expression of steroidogenic enzymes involved in androgen production: 3β‐hydroxysteroid dehydrogenase/Δ5‐Δ4 isomerase (3β‐HSD), cytochrome P450 17α‐hydroxylase/17,20‐lyase (CYP17) and 17β‐hydroxysteroid dehydrogenase type 1 (17β‐HSD1) or type 3 (17β‐HSD3) in the foetal porcine gonads. Pregnant gilts were injected with anti‐androgen flutamide (for seven days, 50 mg/day/kg bw) or corn oil (control) starting at 83 (GD90) or 101 (GD108) gestational day. To assess 3β‐HSD, CYP17 and 17β‐HSD1 or 17β‐HSD3 expression, real‐time PCR and immunohistochemistry were performed. In testes from flutamide‐treated foetuses, increased 3β‐HSD and CYP17 mRNA expression was observed in the GD90 group, while decreased 3β‐HSD and 17β‐HSD3 mRNA expression and increased CYP17 mRNA expression were found in the GD108 group. CYP17 and 17β‐HSD3 were localized in Leydig cells. Following flutamide administration, the intensity of CYP17 immunostaining was higher in both treated groups, while 17β‐HSD3 intensity was lower in the GD108 group. In ovaries from flutamide‐treated foetuses in the GD90 group, mRNA level for 3β‐HSD was elevated, but it was diminished for CYP17 and 17β‐HSD1. In the GD108 group, flutamide treatment led to lower mRNA level for 3β‐HSD but higher for CYP17. 3β‐HSD was found in granulosa cells, while CYP17 was localized within egg nests and oocytes of forming follicles. Following flutamide treatment, the intensity of 3β‐HSD and CYP17 immunostaining was higher in the GD90 and GD108 groups, respectively. Immunohistochemical staining for 3β‐HSD was restricted to the ovary. Concluding, diminished androgen action in the porcine foetal gonads during late gestation induces changes in steroidogenic enzymes expression, which may led to changes in gonadal function. However, it seems that androgens exert diverse biological effects depending on the gestational period.     

Effects of dietary β‐(1,3)(1,6)‐D‐glucan supplementation on growth performance,intestinal morphology and haemato‐immunological profile of mirror carp (Cyprinus carpio L.)

In recent years, aquaculture research has focused on probiotics, prebiotics, and β‐glucans, in order to improve health status and growth performance. Information regarding the effects of β‐glucan on growth performance and intestinal immunity of mirror carp (Cyprinus carpio L.) is scarce. An experiment was therefore conducted to investigate the effects of a yeast β‐glucan preparation (MacroGard^®) on growth performance, intestinal morphology and haemato‐immunological indices of mirror carp. Carp (initial weight 11.1 ± 0.0 g) were fed highly purified diets supplemented with 0% (control), 0.1%, 1% or 2% MacroGard^® for 8 weeks. Fish fed diets containing 1% and 2% MacroGard^® showed significant improvements in weight gain, specific growth rate and feed conversion ratio compared to fish fed both the control and the 0.1% MacroGard^® containing diet. Histological appraisal of the intestine showed a significantly higher infiltration of leucocytes into the epithelial layer of fish fed diets supplemented with 1% and 2% MacroGard^® in the anterior intestine compared to fish fed the control and 0.1% MacroGard^® diet. This effect was not observed in the posterior intestine. There were no significant differences in the intestinal absorptive surface area and number of goblet cells in either intestinal region. At the end of the experiment, the haematological status of the fish was examined. Compared to control fed fish, the haematocrit value was significantly elevated in fish fed the 2% MacroGard^® diet. Furthermore, the blood monocyte fraction was significantly higher in fish fed the 1% and 2% MacroGard^® diets. No significant changes were observed in the other blood parameters assessed. The present study shows that high dietary β‐glucan inclusion increases growth performance without detrimental effects on the health indicators assessed. Increased intraepithelial leucocytes in the anterior intestine may indicate a localized immune response; no detrimental effects on intestinal morphology were observed.     

Effect of D‐mannitol on nitrogen retention,fiber digestibility and digesta transit time in adult rabbits

The aim of the current study was to elucidate the effect of gastrointestinal retention time of digesta on fiber digestibility in adult rabbits fed indigestible, but fermentable, sugar D‐mannitol. Six adult rabbits were fed alternately a commercial diet containing 5% glucose and a diet containing D‐mannitol. Total feces and urine were collected during the experimental period. Nitrogen (N) balance, digestibility of nutrients, and gastrointestinal mean retention time (MRT) were measured. The results indicated that urinary excretion was significantly lowered, whereas N retention and N accumulation rates were significantly increased in the D‐mannitol group compared with the glucose group (P < 0.05). However, fecal N excretion was unaffected. Absorption of crude ash (CA) and acid detergent fiber (ADF) digestibility were significantly higher in the D‐mannitol group compared with the glucose group (P < 0.05). The addition of D‐mannitol to the diet did not affect the MRT of liquid digesta, but increased the MRT of solid digesta compared with the glucose group (P < 0.05). These results suggest that the addition of D‐mannitol to the diet stimulates cecal bacterial growth, thereby increasing N utilization and digesta retention time.     

The Relationship Among Vitamin C, β‐carotene,Vitamin A,Progesterone and Oestradiol 17‐β Concentrations in Plasma and Cyst Fluid of Holstein Cows with Ovarian Cyst

The aims of this study were to determine the concentrations of the progesterone, oestradiol‐17‐β, vitamin A, C and β‐carotene in plasma and cyst fluid and to relate these values with cystic diameter and membrane thickness of Holstein cattle with ovarian luteal cyst. 1650 Holstein cows were examined for the presence of the ovarian cyst and luteal and follicular cystic ovaries were obtained following slaughtering in personal slaughterhouse in Konya‐Turkey. 15 Luteal and 15 follicular cystic ovaries were distinguished by rectal palpation and by post mortem ultrasonographic examination. Plasma and cyst fluid, hormone and vitamin analyses were carried out by EIA method and spectrophotometric measurement respectively. Although there was no relationship between β‐carotene and vitamin A in plasma and cyst fluid of both cyst type and hormone concentrations, the vitamin C concentration of cyst fluid was found significantly higher in luteal cyst than in follicular cyst. Moreover, there is a positive correlation among values of the vitamin C concentrations of cyst fluid and cystic membrane thickness, plasma and the cyst fluid progesterone concentrations, but there is a negative correlation among the vitamin C concentrations of cystic fluid and oestradiol 17β levels of plasma and cyst fluid. In conclusion, vitamin C concentration of cyst fluid supported ultrasonographic and endocrinologic findings. Also, it can be postulated that vitamin C is probably effective on progesterone synthesis in the luteal tissue of cyst.     

The growth of the canine glioblastoma cell line D‐GBM and the canine histiocytic sarcoma cell line DH82 is inhibited by the resveratrol oligomers hopeaphenol and r2‐viniferin

Vineatrol^®30 is a grapevine‐shoot extract, which contains resveratrol as well as considerable amounts of so‐called resveratrol oligomers such as hopeaphenol and r2‐viniferin. In this study, we analysed whether the two above‐mentioned resveratrol oligomers were able to inhibit the growth of the canine glioblastoma cell line D‐GBM and the canine histiocytic sarcoma cell line DH82, compared their potency to inhibit tumour cell growth with that of resveratrol and determined whether the induction of apoptosis via caspase 9 and 3/7 activation underlies the tumour cell growth‐inhibiting effect of hopeaphenol and r2‐viniferin. Vineatrol^®30, resveratrol, hopeaphenol and r2‐viniferin inhibited the growth of D‐GBM and DH82 cells in a concentration‐dependent manner, whereby hopeaphenol and r2‐viniferin were more potent than resveratrol itself in inhibiting the growth of the canine tumour cell lines. Moreover, the anti‐proliferative effect of both resveratrol oligomers in D‐GBM cells is based on their capacity to induce caspase 9 and 3/7 activation.     

Nuclear Replacement of In Vitro‐Matured Porcine Oocytes by a Serial Centrifugation and Fusion Method

The objective of the present study was to establish a method for nuclear replacement in metaphase‐II (M‐II) stage porcine oocytes. Karyoplasts containing M‐II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro‐matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona‐free M‐II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2–77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2–32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0–15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri‐Fusion) is an effective method for producing M‐II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.     

Follicular Dynamics,Interval to Ovulation and Fertility After AI in Short‐Term Progesterone and PGF2α Oestrous Synchronization Protocol in Sheep

The study was aimed to assess the influence that short‐term progesterone treatments have on follicular dynamics, oestrus and ovulation in sheep. The treatment was tested thereafter in a field trial to assess its fertility after AI with fresh semen. In a first experiment, 12 ewes without CL were grouped to receive a new (n = 6) or used CIDR (n = 6) for 7 days and blood samples were obtained to follow plasma progesterone profiles. In a second experiment, 39 cycling ewes were synchronized by a 7‐day P4+PGF2α protocol using a new (n = 20) or a 7‐day used CIDR (n = 19). Half of both groups received 400 IU eCG and half remained untreated as controls. Ultrasound ovarian examination and oestrous detection were used to compare follicular dynamics, oestrus and ovulation in both groups. In a third experiment, 288 ewes in 3 farms were synchronized by the short‐term P4+PGF2α+eCG protocol and ewes were AI with fresh semen 24 h after oestrous detection. Lambing performance was used to test the fertility of the treatment. In Experiment 1, ewes with new inserts presented higher P4 concentration than ewes with used inserts throughout the sampling period (p < 0.05) and exhibited a P4 peak at days 1‐2 of the treatment that was not observed in ewes with used inserts. In Experiment 2, ewes treated with new and used inserts show similar ovarian and behavioral traits (p > 0.10). However, ewes treated with eCG show shorter interval to oestrus (p = 0.004) and tend to have larger mature CL (p = 0.06). In Experiment 3, oestrous presentation and lambing performance after AI with fresh semen was considered normal compared to published results. Results suggest that the oestrous synchronization protocol based on P4+PGF2α allows little control of follicular dynamics without compromising fertility after AI with fresh semen provided that eCG is added at the end of the treatment.