Sperm Pretreatment with Dithiothreitol Increases Male Pronucleus Formation Rates After Intracytoplasmic Sperm Injection (ICSI) in Swamp Buffalo Oocytes

Failure of male pronucleus formation has hampered the success of intracytoplasmic sperm injection (ICSI) in swamp buffalo. The aim of the present study was to improve male pronucleus formation by pretreating sperm with various chemicals before ICSI. In Experiments1 and 2, sperm were treated according to one of the following protocols: (1) 0.1% Triton-X 100 (TX) for 1 min, (2) 10 µM calcium ionophore (CaI) for 20 min, (3) freezing and thawing (FT) without any cryoprotectant, or (4) no treatment (control). These sperm treatment groups then either did or did not receive additional sperm treatment with 5 mM dithiothreitol (DTT) for 20 min. Acrosomal integrity (Experiment 1) and DNA fragmentation (Experiment 2) were evaluated in the sperm before ICSI. In Experiment 3, oocytes matured in vitro were subjected to ICSI using pretreated sperm as described above and then were cultured either with or without activation. The TX- and CaI-treated sperm caused an increase in the number of acrosome-loss sperm, whereas the FT treatment and control increased the proportion of acrosome-reacted sperm (P<0.05). The DNA fragmentation did not differ among treatments (P>0.05). At 18 h post-ICSI, pronucleus (PN) formation was found only in activated oocytes. The majority of the activated ICSI oocytes contained intact sperm heads. Normal fertilization was observed in the CaI and FT treatment groups and control group when sperm were treated with DTT before ICSI. In conclusion, DTT treatment of sperm with reacted acrosomes before ICSI together with activation of the ICSI oocytes is important for successful male pronucleus formation.     

Effect of dexmedetomidine vs. acepromazine–methadone premedication on limb to lung circulation time in dogs

The study compared limb-to-lung circulation times (CT) in dogs under general anaesthesia after premedication with dexmedetomidine (DEX) or acepromazine–methadone (ACE–M). Healthy male and female dogs (n = 20) were randomly assigned to receive acepromazine 0.04 mg/kg and methadone 0.2 mg/kg intramuscularly (IM), or DEX 0.01 mg/kg IM. Anesthesia was induced with propofol and maintained with isoflurane at similar concentration in both groups. Mechanical ventilation was started immediately (20 breaths/min; inspiratory to expiratory ratio 1:2) and tidal volume was adjusted to achieve an end-tidal CO₂ concentration (PE’CO₂) of between 3.9 and 5.3 kPa. Ten minutes later arterial blood gas was analyzed and baseline data recorded for 3 minutes. A single dose of sodium bicarbonate 0,5 mEq/kg was administered intravenously over 10 s starting with inspiration. Limb-to-lung CT was defined as the time interval between the start of bicarbonate injection and the recording of the highest PE’CO₂.Following bicarbonate administration, PE’CO2 increased, and then rapidly decreased to baseline in both groups. CT was shorter in the ACE–M group (20 ± 2.3 vs. 27 ± 5.1 s). Bodyweight was higher in the ACE–M group (30.6 ± 3.9 vs. 23.3 ± 6.8 kg). Mean arterial blood pressure was higher in the DEX group (92 ± 9 vs. 73 ± 7 mm Hg) but premedication with DEX significantly prolonged CT compared to premedication with ACE–M.     

Naloxone increases maturation rate and ratio of inner cell mass to total cells in blastocysts in pigs

The purposes of the present study were to examine the effect of naloxone, a mu‐opioid receptor (MOR) antagonist, on porcine oocyte maturation and embryo development. MOR gene was expressed in germinal vesicle (GV) and metaphase II (M‐II) porcine oocytes, one‐, four‐cell stage embryos and blastocysts. In blastocysts, MOR gene was mainly expressed in inner cell mass (ICM) cells. Supplementation of 10^?8 mol/L naloxone in in vitro maturation (IVM) medium increased the maturation rate (P < 0.05). However, 10^?4 mol/L naloxone reduced the maturation rate (P < 0.05) compared with the control. The presence of naloxone during IVM had no effects on fertilization status and subsequent embryonic development after in vitro culture (IVC). The addition of 10^?3 mol/L dibutyryl cyclic adenosine monophosphate (dbcAMP), and 10^?8 mol/L naloxone together into IVM medium increased nuclear maturation (P < 0.05) compared with the addition of either dbcAMP or naloxone alone. Supplementation with naloxone in IVC medium did not improve embryonic development. However, at the concentrations of 10^?6 mol/L and 10^?8 mol/L, naloxone increased the ratio of ICM to total cells in blastocysts (P < 0.05). In conclusion, at low concentration, naloxone increases maturation rate and the ratio of ICM to total cells in blastocysts. Naloxone and cAMP have a synergistic effect on oocyte maturation.     

Resistance to velogenic Newcastle disease virus in leghorn chickens by use of prophylactic lymphokines

A group of 1-day-old commercial leghorn chickens was prophylactically treated with lymphokines obtained from lymphocyte cultures of chickens previously infected with Salmonella enteritidis (S. enteritidis-immune lymphokines [SE-ILK]) with the objective to investigate the effect of SE-ILK on development of Newcastle disease (ND) infection caused by Chimalguacan strain, a Mexican velogenic ND virus (vNDV). Clinical signs, histologic lesions, and hemagglutination-inhibition (HI) serum titers were compared with four other groups, namely, chickens without SE-ILK treatment with virus challenge; with SE-ILK without virus challenge; with nonimmune lymphokine (NILK) treatment and virus challenge; with lymphokine treatment and no virus challenge. SE-ILK was administered intraperitoneally in a dose of 0.5 ml/chicken and was followed 30 min later with the challenge of vNDV in a dose of 10(7.6) 50% embryo lethal dose/ml per bird. Birds were observed during 21 days of postchallenge. Detection of histologic changes and virus isolation procedures were carried out on the third, seventh, 14th, and 21st postinoculation days. HI tests were performed first before treatment and later on the days of histologic sample collection except on the third postinoculation day. Results showed that SE-ILK administration conferred resistance to the chickens because: 1) it significantly diminished the severity of ND infection by inhibiting appearance of clinical signs (P < 0.001), lesions (P < 0.005), and histopathologic changes (P < 0.005); 2) it decreased vNDV isolation rate from the organs (P < 0.001), and 3) it potentialized and even accelerated (P < 0.005) primary immune response by antibodies in the presence of vNDV.     

Effect of centrifugation treatment before vitrification on the viability of porcine mature oocytes and zygotes produced in vitro

The aim of the present study was to investigate the effects of centrifugation pretreatment on the viability and nuclear status of porcine in vitro matured (IVM) oocytes and on the developmental competence of in vitro fertilized (IVF) oocytes (zygotes) after cryopreservation by vitrification (Solid Surface Vitrification; SSV). Mature oocytes having the first polar body after IVM and zygotes having the second polar body at 10 h after IVF were centrifuged at 10,000 x g at 37 C for 20 min and then subjected to SSV. Their viability was evaluated by morphological appearance and fluorescein diacetate staining. The nuclear status of oocytes was evaluated 6 h after vitrification. The developmental ability to the blastocyst stage of vitrified zygotes was evaluated after 6 days of in vitro culture. Although centrifugation did not damage the oocytes directly, it drastically reduced the rate of live oocytes after SSV. The rates of vitrification-induced parthenogenetic activation were similar in both centrifuged and non-centrifuged oocytes (42.4 and 47.4%, respectively). Centrifugation had no significant effects on the viability of pronuclear oocytes. The development of vitrified zygotes to the blastocyst stage was significantly lower than that of the control irrespective of centrifugation pretreatment. There was no difference in the cleavage and blastocyst rates between the control and centrifuged zygotes after vitrification. There was also no difference in the total cell numbers of blastocysts between the control and centrifuged zygotes irrespective of vitrification. These results reveal that, in IVM porcine oocytes, centrifugation pretreatment is highly detrimental to cryotolerance; however, in zygotes, it has only a slight effect on viability and does not alter the developmental competence of surviving zygotes.     

Effect of medium additives during liquid storage on developmental competence of in vitro matured bovine oocytes

Our aim was to improve the developmental competence of bovine oocytes during their liquid storage by using additives. In vitro matured oocytes were stored for 20 h at 25°C in HEPES buffered TCM 199 medium (base medium). After storage, in vitro embryo development after in vitro fertilization was compared to those of non‐stored (control) ones. Addition of 10% (v/v) newborn calf serum or 10.27 mmol/L pyruvate alone to the base medium did not improve blastocyst formation rates in stored oocytes; however, their simultaneous addition significantly improved the rate compared with those stored in base medium (P < 0.05). Supplementation of the holding medium with dithiothreitol (DTT) at any concentrations did not improve embryo development from stored oocytes. Although supplementation with cyclosporine A (CsA) significantly reduced apoptosis and membrane damage rates during storage, it did not improve the developmental competence of oocytes. 1,2‐bis(2‐aminophenoxy) ethane N,N,N’,N’‐tetraacetic acid tetrakis‐acetoxymethyl ester and ruthenium red had no effect on oocyte apoptotic rates. Blastocyst formation rates in all stored groups remained significantly lower than that of the control. In conclusion, pyruvate and serum had a synergic effect to moderate the reduction of oocyte quality during storage, whereas mitochondrial membrane pore inhibitor CsA and the antioxidant DTT did not affect their developmental competence.     

Assessment of distribution of ventilation and regional lung compliance by electrical impedance tomography in anaesthetized horses undergoing alveolar recruitment manoeuvres

Objective

To examine changes in the distribution of ventilation and regional lung compliances in anaesthetized horses during the alveolar recruitment manoeuvre (ARM).

Antifungal activity of Aloe vera leaves

Aloe vera fresh leaves hydroalcoholic plant extract was tested against the mycelial growth of Botrytis gladiolorum, Fusarium oxysporum f.sp. gladioli, Heterosporium pruneti and Penicillium gladioli on Czapek-agar medium. The minimum fungicidal concentration (MFC) varied between 80 and 100 microl/ml, depending on the fungal species.