Expression of the μ Opioid Receptor and Effects of the Opioid Antagonist Naloxone on In Vitro Maturation of Oocytes Recovered from Anoestrous Bitches

The μ-opioid receptor (MOR) is expressed in bovine, human, equine and canine oocytes, and in seasonal breeders, it is expressed with higher intensity during the anoestrous phase. Supplementation of in vitro maturation (IVM) medium with opioid agents, agonists or antagonists, was shown to affect oocyte maturation in several species such as rat, bovine and equine. This study reports the effects of supplementing IVM medium with naloxone (Nx), an opioid antagonist, on nuclear and cytoplasmic maturation rate of oocytes recovered from anoestrous bitches. Cytoplasmic maturation was examined in terms of mitochondrial (mt) distribution. In order to confirm the receptor-mediated action of Nx, in oocytes of anoestrous bitches, MOR expression was analyzed by Western blot. Cumulus–oocyte complexes, recovered from the ovaries of bitches in anoestrous, were cultured in vitro and Nx was added at the concentrations of 1 × 10⁻⁶, 1 × 10⁻⁸ and 1 × 10⁻¹⁰  m . The rate of oocytes resuming meiosis after culture in presence of 1 × 10⁻⁶  m Nx (29%) was significantly higher than that of oocytes of control group (12%; p < 0.05). However, treatment with Nx did not affect mt distribution pattern. In denuded oocytes and in corresponding cumulus cells, a doublet of 65 and 50 kDa was observed. We conclude that, in oocytes of anoestrous bitches, MOR is expressed and Nx significantly improves nuclear maturation rate. Further studies should be performed to elucidate the expression of other opioid receptors, such as δ and κ, and possible interactive effects of their antagonists on canine oocyte maturation.     

Standing Laparoscopic Peritoneal Flap Hernioplasty of the Vaginal Rings does not Modify the Sperm Production and Motility Characteristics in Intact Male Horses

Laparoscopic hernioplasty techniques have been developed in the recent years to avoid the recurrence of inguinal hernias and to spare the testicles for breeding purposes in stallions. However, there have been no previous comprehensive and systematic studies of the reproductive outcomes and prognoses for stallions after inguinal hernioplasty. Therefore, the objective of this study was to assess the possible effects of one of these techniques (standing laparoscopic peritoneal flap hernioplasty) on the sperm production and motility characteristics of six healthy stallions that received this procedure based on 1‐year follow‐ups. There were no significant differences in the measured sperm variables (assessments based on the DSO, MOT, PMOT, VSL, VCL and VAP) during 1‐year follow‐ups.     

Milk Progesterone Profile at and after Artificial Insemination in Repeat-Breeding Cows: Effects on Conception Rate and Embryonic Death

The aim of this study was to investigate whether the skim milk progesterone concentrations at artificial insemination (AI) and day of rise of post-ovulatory progesterone concentration thereafter affect the conception and embryonic death rates in repeat-breeding cows. Milk samples were obtained from 96 repeat-breeding cows that failed to conceive to three or more AIs. The samples were taken from the cows at the day of AI and three times/week until day 45 post-AI. Skim milk was obtained after centrifugation and used for progesterone assay. The cows with a progesterone concentration more than 0.5 ng/ml at AI showed a significantly higher incidence of late embryonic death than those having a progesterone concentration<0.5 ng/ml at AI (p<0.01). As the progesterone level at insemination rose, conception rate declined. A negative correlation was shown between conception rate and skim milk progesterone level at AI. Of 56 cows showing a rise of progesterone to 1 ng/ml or more within 6 days after AI, 28 cows (50%) conceived. On the contrary, only eight of 39 cows (20.5%) conceived when the progesterone rose up to 1 ng/ml after day 6 post-AI. We concluded that increased progesterone concentration at the time of AI and delayed rise of progesterone post-AI might lead to decrease in fertility in repeat-breeding cows.     

Pathological Conditions of the Reproductive Organs of Male Stray Dogs in the Tropics: Prevalence, Risk Factors, Morphological Findings and Testosterone Concentrations

The objective of this study was to estimate the prevalence of and risk factors for pathological conditions of the reproductive organs in stray dogs under tropical conditions. Three hundred and eighteen dogs were examined post-mortem in the period from 1 July 2002 to 30 June 2003. Before killing, a blood sample (from the cephalic vein) for testosterone assay was taken. Pathological conditions of the reproductive organs were found in 135 of the dogs (42.5%) and in 175 of the testes (64.8%). The most frequent pathologies found were testicular degeneration, cryptorchidism, testicular hypoplasia and testicular tumours (in 15.1%, 6.6%, 6.6% and 5.4% of the dogs and 15.1, 4.6, 6.0 and 3.5 of the testes, respectively). Transmissible venereal tumour (TVT) was seen in 5.4% of the dogs. Testicular degeneration was more common in old dogs and underweight dogs (p < 0.05). Testicular tumours were 14.3 times more common in cryptorchid dogs. Age was another important factor for the development of testicular tumours (p < 0.05). Lower levels of testosterone concentration (p < 0.05) were observed in dogs with advanced testicular degeneration (0.7 +/- 0.8 nM), dogs with hypoplastic testicles (0.8 +/- 0.9 nM) and dogs with one degenerated and one retained testis or with bilateral cryptorchidism (1.2 +/- 0.9 nM) compared to dogs with one or two normal testes (7.0 +/- 5.5 nM). Testicular volume and weight were significantly lower in degenerated, hypoplastic and retained testes compared with the contralateral normal testis. Some spermatogenic activity was found in three of the retained testes, producing oligozoospermic smears with a high percentage of sperm abnormalities. No comparable epidemiological data about male pathological conditions of the reproductive organs in the dog is available. The prevalence found in this study, yet, appears high.     

Effect of Complex Vertebral Malformation on Luteal Function in Holstein Cows During Oestrous Cycle and Early Pregnancy

The reason why cows carrying the mutation of complex vertebral malformation (CVM) show poor reproductive capability although they carry only one mutant allele is still not fully understood. Monitoring the progesterone profiles during oestrous cycle and early pregnancy in carrier cows might help explain their lowered reproductive capability. Progesterone concentration was measured in 19 CVM carrier cows and 21 control cows during oestrous cycle and early pregnancy. Milk samples were collected from all cows starting on the day of artificial insemination until day 45 post‐AI. Progesterone was measured in skim milk using enzyme‐linked immunosorbent assay (ELISA). Progesterone concentration was significantly reduced on day 7 (p < 0.05) and day 9 (p < 0.01) post‐insemination in conceived CVM carrier cows when compared with that in control conceived cows. The mean progesterone concentration during early pregnancy was significantly lower (p < 0.05) in conceived cows with CVM than that of control cows in the same period. However, the mean progesterone concentration did not differ significantly (p = 0.072) in CVM cows that showed fertilization failure or embryonic death than that of control cows. Additionally, of 13 conceived control cows, eight cows (61.5%) showed normal luteal function. In contrast, of nine conceived CVM cows, only four cows (44.4%) showed normal luteal function. The conception rate was 47.4% in CVM carrier cows and 61.9% in control cows, but this difference did not reach significance. In conclusion, progesterone concentration might be lowered during early pregnancy in conceived CVM cows compared with that in control cows.     

Role of Cumulus Cells on In Vitro Maturation of Canine Oocytes

The objectives of the present study were to investigate the relationship between the morphological status of cumulus cells surrounding canine oocytes after maturation culture and the meiotic stage of the oocytes. In addition, the effect of the removal of cumulus cells from canine cumulus-oocyte complexes (COCs) during maturation culture on their meiotic competence was examined. Canine COCs were collected from bitches at the anoestrous and dioestrous stages and only COCs with >110 microm in vitelline diameter were cultured in medium 199 with 10% canine serum for 72 h. In the first experiment, the relation between the morphological status of cumulus cells surrounding oocytes cultured for 72 h and their meiotic stages was examined. At the end of maturation culture, the proportions of intact, partially nude and completely nude oocytes were 65.2%, 22.9% and 11.9%, respectively. The proportion of maturation to metaphase II of completely nude oocytes was highest among the oocytes with different morphological status of cumulus cells. In the second experiment, the cumulus cells were partially or completely removed from COCs at 48 h after the start of maturation culture and the oocytes were cultured for a further 24 h. The proportion of oocytes reaching metaphase II in the completely denuded oocytes was significantly higher than that in the control oocytes without the removal treatment of cumulus cells. The results indicate that morphological status of cumulus cells surrounding oocytes may be related to the nuclear maturation of canine oocytes, and the removal of cumulus cells from COCs during maturation culture can promote the completion of oocyte meiotic maturation.     

Effect of different mini‐volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer‐assisted semen analyzer

Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 10⁶ sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm^®. A single layer of 40% PureSperm^® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm^® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm^® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm^® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm^® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.     

Pattern of bombardment-produced radionuclides in rock 10017 and in lunar soil

A large number of radionuclides have been measured as a function of depth in lunar rock 10017 and in bulk fines. Data are reported on (10)Be, (22)Na, (26)Al, (36)Cl, (49)V, (53)mn, (54)Mn (55)Fe, (56)Co, (57)Co, and (59)Ni and on upper limits for (46)Sc, (48)V, (51)Cr, and (60)Co. The results for several nuclides show striking evidence of excess surface production attributable to solar flare particles. Data for short-lived species, (56)Co, (57)CO, (54)Mn, (55)Fe, and (22)Na, appear consistent with fluxes from known recent events. Long-lived species demonstrate the existence of solar flare protons and alphas at least for the last 10(5) to 10(6) years, at fluxes comparable to those now observerved.     

Magnetic field effect on picosecond electron transfer

The recombination dynamics of a transition metal redox system monitored by femtosecond pump-probe spectroscopy are shown to be sensitive to high magnetic fields at times shorter than 10 picoseconds. The effect, based on coherent population beats of different spin states, is quantitatively accounted for and allows direct access to spin relaxation rates far beyond the time resolution of the fastest electron paramagnetic resonance technique. The presence of this ultrafast magnetic field effect helps in understanding complex reaction schemes in transition metal chemistry, which occur in a wide range of fields, such as bioinorganic chemistry and catalysis.