Sensitive and specific polymerase chain reaction detection of Toxoplasma gondii for veterinary and medical diagnosis.

A polymerase chain reaction (PCR) method was developed for the detection of Toxoplasma gondii. A universal- and a T. gondii-specific primer was used to amplify a region of the small subunit ribosomal RNA gene. This approach allows for a theoretical detection limit of 0.01 zoite of T. gondii per sample assayed. Experiments showed that this PCR method could detect 0.1 pg of T. gondii DNA, which represents about one organism. Polymerase chain reaction tests using DNAs of cat, dog, swine, cattle, human, Sarcocystis cruzi, Eimeria ahsata, E. vermiformis, and Escherichia coli indicated no cross-reaction with nucleic acids of hosts, related coccidia, or bacteria. Data on the sensitivity and specificity suggest that this PCR assay could be extremely useful for the diagnosis of toxoplasmosis in human and veterinary medicine, as well as for food safety surveys.     

Modulation of Glycolysis and the Pentose Phosphate Pathway Influences Porcine Oocyte In Vitro Maturation

Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus–oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose‐dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6‐AN) and the physiological (NADPH) inhibitors of PPP induced a dose‐dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.     

Embryo Production in Dogs: from In Vitro Fertilization to Cloning

Increased availability of canine embryos would be desirable to develop research and to apply assisted reproductive technologies in the treatment of infertility and in the improvement of reproductive performances in valuable Canids, both domestic and non-domestic. Embryo production through in vitro fertilization and nuclear transfer has been technically achieved in the dog, and the transfer of cloned embryos has recently resulted in the birth of puppies. However, the efficiency of these technologies is still very limited. This is mainly because of the peculiar characteristics of the canine oocyte and the lack of its full acquisition of developmental competence in vitro. This paper discusses the latest results and aspects on which further research should be focused to provide advances in the field.     

Tris-Lecithin Extender Supplemented With Antioxidant Catalase for Chilling of Canine Semen

The aims were to evaluate the suitability of a non-commercial TRIS-lecithin (LC) extender and the effect of different concentrations of catalase (CAT) on motility, capacitation status (Chlortetracycline-assay) and zona pellucida (ZP) binding capacity of canine spermatozoa stored at +5°C for 4 days. The sperm-rich fractions of the ejaculates of four stud dogs were divided into four aliquots. After centrifugation, sperm pellets were diluted (200 × 10⁶ sperm/ml) in TRIS buffer, citric acid, fructose, antibiotics, supplemented with 20% egg yolk (TRIS-EY) or 0.04% soybean lecithin (TRIS-LC) with CAT (150 or 450 UI/ml) or without CAT, and then preserved at 5°C for 4 days. The results showed that LC is a valid alternative to EY for chilling canine semen, as similar rates of motility, number of uncapacitated spermatozoa and of spermatozoa binding the oocyte ZP were obtained in semen chilled in TRIS-LC or TRIS-EY. Different concentrations of CAT in a TRIS-LC based extender did not improve the quality of semen after chilling. However, a concentration of 150 UI/ml CAT resulted in an increased number of spermatozoa bound to the oocyte ZP, after 4 days of chilling when compared to semen chilled with TRIS-EY and TRIS-LC. In conclusion, an animal protein-free extender with soybean LC, as a replacement of EY, is suitable for 4 days chilling of canine spermatozoa, but the addition of CAT does not improve general semen quality except for a slight effect on sperm-ZP binding.     

Meiosis Resumption of Canine Oocytes Cultured in the Isolated Oviduct

The aim of this study was to investigate the effects of culture in isolated oviducts relative to meiotic maturation, the time required to resume meiosis and the viability of the canine oocytes. For this purpose, cumulus–oocyte complexes and isthmus–ampullar tracts of the oviducts were collected from bitches undergoing ovariohysterectomies and destined to two experiments of culture. In experiment 1, the oocytes were cultured for 24 or 30 h: (1) in 100 μl drops under oil; (2) on the mucosal epithelium of the open oviducts; (3) in the ligated oviducts. In experiment 2, oocytes were cultured in the ligated oviduct for 24, 30 and 48 h. A group of control oocytes was not cultured (0 h). The results showed that within 30 h of culture, a higher proportion of oocytes (p < 0.001) resumed meiosis in the ligated oviduct (63.8%) than in drop (20.4%) or in the open oviduct (27.1%). Moreover, 24 and 30 h of culture assured higher proportions of meiosis resumption than 48 h (69.2 and 59.1% vs 35.8%, p < 0.005). Oocyte resumption of meiosis was mainly determined by oocytes at meiotic stages preceding metaphase I, while stages between metaphase I and II in the ligated oviduct ranged between 12.5 and 31.9%. The extension of the culture time up to 48 h in the oviduct increased oocyte degeneration significantly (59.3%, p < 0.0001) compared with 24 and 30 h (18.7 and 27.3%, respectively) and the oviductal epithelium showed nuclear picnosis and degeneration following culture. The present study suggests that the close physical interaction between the canine oocytes and the oviductal tract positively affects oocyte maturation, and meiosis is resumed within 30 h of culture. Moreover, the oocyte survival is better preserved within 30 h in the ligated oviduct compared with the conventional culture in drop or to the culture in the open oviduct, but the ligated oviduct does not assure viability of the oocytes up to 48 h of culture.     

Effects of Superovulation with oFSH and Norgestomet/GnRH‐Controlled Release of the LH Surge on Hormone Concentrations,and Yield of Oocytes and Embryos at Specific Developmental Stages

The objective of this study was to evaluate a new superovulation procedure with oFSH after temporary suppression of the endogenous LH surge by norgestomet followed by administration of GnRH, to collect bovine oocytes and embryos at specific developmental stages. Since 1999, our research group applies this superovulation procedure with controlled release of the endogenous LH surge. The objective of this study is to verify if this procedure is reliable for collection of oocytes and embryos at specific time points of development and if it produces a sufficient number of both oocytes and embryos of good quality. This procedure was validated regarding to hormonal characteristics, superovulatory response and both oocyte and embryo yield at different times of in vivo development. The results demonstrate that the procedure used to control the occurrence of the pre‐ovulatory LH surge was effective in 92% of the animals (n = 238) and even in 99% of the animals the oocytes and embryos were collected at the intended stage of development. The superovulatory response and both oocyte, embryo yield and quality were similar to the average yield in Europe reported by Association Européenne de transfert embryonnaire (AETE). In conclusion, this superovulation procedure provides a valid tool to collect oocytes and embryos at specific time points of development.     

Incidence and clinical significance of sesamoid disease in rottweilers.

A group of 55 rottweiler pups was studied from three to 12 months old to assess the incidence and clinical significance of disease involving the palmar metacarpal sesamoid bones. The results of physical examination were correlated with clinical signs of lameness and the results of radiographic examination of the forefeet. Twenty-one dogs became lame during the study and in 12 of them the lameness was attributable to sesamoid disease. However by 12 months of age, the incidence of sesamoid disease as assessed by radiographic changes in the sesamoid bones was 73 per cent (30 of 41 dogs). Six of the 12 dogs which were lame owing to sesamoid disease got better without specific treatment. It was concluded that sesamoid disease can result in clinical lameness in young rottweilers, but that subclinical disease is common.