High developmental rates of mouse oocytes cryopreserved by an optimized vitrification protocol: the effects of cryoprotectants, calcium and cumulus cells

Unfertilized oocytes are one of the most desired germ cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. The aim of the present study was to improve vitrification of mouse oocytes. First, the effects of calcium and cryoprotectants, dimethyl sulfoxide and ethylene glycol (EG), in vitrification medium on survival and developmental ability of vitrified oocytes were evaluated. Oocytes were vitrified by a minimal volume cooling procedure using different cryoprotectants. Most of the vitrified oocytes were morphologically normal after warming, but their fertility and development were low independently of calcium and cryoprotectants. Second, the effect of cumulus cells on ability of oocytes to be fertilized and develop in vitro was examined. The fertility and developmental ability of denuded oocytes (DOs) after IVF were reduced compared with cumulus-oocyte complexes (COCs) both in fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop to the 2-cell and blastocyst stages than those of vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate equivalent to the rate obtained with IVF using fresh COCs. Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and EG retain their developmental competence. These findings will contribute to improve oocyte vitrification in not only experimental animals but also clinical application for human infertility.     

Long-lasting enhancement of CYP activity after discontinuation of repeated administration of phenobarbital in dogs

We investigated how long in vivo hepatic cytochrome P450 (CYP) activity is enhanced even after discontinuation of repeated oral administration of phenobarbital (PB) in dogs using antipyrine clearance, which reflects hepatic CYP activity. A single antipyrine (5 mg/kg) was administered intravenously before and 34 days after the repeated oral administration of PB (5 mg/kg, bid) and 2, 4, 6, and 8 weeks after the discontinuation of PB in 5 dogs. Antipyrine clearance was increased by the repeated administration of PB, and remained increased 2 and 4, but not 6 and 8 weeks after the discontinuation of PB. The result suggests that hepatic CYP activity was enhanced by the repeated administration of PB, and this enhancement may last for at least 4 weeks even after its discontinuation.     

Localization of endothelin receptor subtype-A in the testis of rats, dogs, and monkeys

In order to evaluate the physiological roles of the testicular endothelin (Edn) signaling via Edn receptor subtype-A (Ednra) in mammals, the localization of Ednra was investigated by in situ hybridization and immunohistochemistry in the testis of rats, dogs, and monkeys. For in situ hybridization, a rat Ednra RNA probe which is highly homologous to the subcloned canine and monkey Ednra (88.7% and 87.9% identical, respectively) was used. Both Ednra mRNA and protein were detected in interstitial cells and cells in the basal compartment of the seminiferous tubules, mainly Sertoli cells, as well as spermatogonia and some early spermatocytes, but not spermatids. The localization pattern of Ednra was exhibited in a same manner among species, indicating that the physiological role of Edn signaling throughout Ednra was maintained in the mammalian testis.     

Exfoliation rate of mammary epithelial cells in milk on bovine mastitis caused by Staphylococcus aureus is associated with bacterial load

The exfoliation rate of mammary epithelial cells (MECs) in milk is affected by physiological, breeding and environmental factors. Little is known about the relationship between the MEC exfoliation into milk and mammary‐infected Staphylococcus aureus (S. aureus) load on bovine mastitis caused by S. aureus. The aim of this study was to investigate the relationship between S. aureus load and the proportion of MEC exfoliation in milk using five substantial bovine mastitis models. In 64 randomly extracted milk samples from udders at 3–21 days after S. aureus infusion, there were various samples with different numbers of S. aureus counts and somatic cell counts. No significant correlations were found between the S. aureus counts and somatic cell count (r = 0.338). In contrast, a significant correlation was noted between S. aureus counts and the proportion of cytokeratin‐positive cells in the milk from the infused udders (r = 0.734, P < 0.01). In conclusion, the increasing MEC exfoliation rate in milk from mastitis udders caused by S. aureus may contribute to reduced milk yield.     

Comparisons among MRI signs,apparent diffusion coefficient,and fractional anisotropy in dogs with a solitary intracranial meningioma or histiocytic sarcoma

Although MRI has become widely used in small animal practice, little is known about the validity of advanced MRI techniques such as diffusion‐weighted imaging and diffusion tensor imaging. The aim of this retrospective analytical observational study was to investigate the characteristics of diffusion parameters, that is the apparent diffusion coefficient and fractional anisotropy, in dogs with a solitary intracranial meningioma or histiocytic sarcoma. Dogs were included based on the performance of diffusion MRI and histological confirmation. Statistical analyses were performed to compare apparent diffusion coefficient and fractional anisotropy for the two types of tumor in the intra‐ and peritumoral regions. Eleven cases with meningioma and six with histiocytic sarcoma satisfied the inclusion criteria. Significant differences in apparent diffusion coefficient value (× 10^?3 mm²/s) between meningioma vs. histiocytic sarcoma were recognized in intratumoral small (1.07 vs. 0.76) and large (1.04 vs. 0.77) regions of interest, in the peritumoral margin (0.93 vs. 1.08), and in the T2 high region (1.21 vs. 1.41). Significant differences in fractional anisotropy values were found in the peritumoral margin (0.29 vs. 0.24) and the T2 high region (0.24 vs. 0.17). The current study identified differences in measurements of apparent diffusion coefficient and fractional anisotropy for meningioma and histiocytic sarcoma in a small sample of dogs. In addition, we observed that all cases of intracranial histiocytic sarcoma showed leptomeningeal enhancement and/or mass formation invading into the sulci in the contrast study. Future studies are needed to determine the sensitivity of these imaging characteristics for differentiating between these tumor types.     

The effect of pretreatment with different doses of GnRH to synchronize follicular wave on superstimulation of follicular growth in dairy cattle

The objective of this study was to evaluate the efficiency of gonadotropin releasing hormone (GnRH) and GnRH doses in synchronizing follicular wave emergence as a pretreatment for superovulation in cattle. Fourteen Holstein-Friesian cows 6 days from estrus were randomly assigned to receive 100 microg (n=4), 50 microg (n=5), or 25 microg (n=5) of GnRH. Superovulation was induced with injections of porcine FSH (pFSH) twice daily, decreasing the dose (total 42 AU) over 5 days beginning 2.5 days after receiving GnRH. On the 7th and 8th injections of pFSH, 750 microg of PGF(2alpha) was also given. With the exception of one cow that was given 50 microg of GnRH, ovulation was induced in all cows from the three groups and the new follicular wave emergence was observed. The total number of follicles for the 25 microg GnRH group was less than that observed for the 100 microg GnRH group (P<0.05), although there were no differences between the 100 microg, 50 microg and 25 microg GnRH groups with respect to the number of preovulatory follicles (>or=10 mm) and CL. The numbers of normal embryos were greater for the 25 microg GnRH group than the 100 or 50 microg GnRH groups (P<0.01); however, the numbers of ova/embryos did not differ significantly between the three groups. These results suggest that 25 microg of GnRH was sufficient to induce ovulation and follicular wave emergence. On day 6 of the estrous cycle, a reduction of the dose of GnRH to synchronize follicular wave emergence as a pretreatment for superstimulation promotes transferable embryos.     

Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis

The vectorial competence of the tick Rhipicephalus sanguineus is discussed in relation to the epidemiology of canine visceral leishmaniasis, taking into account its strict association with dogs and the low indices of natural infection presented by its known vector, the phlebotomine sand fly Lutzomyia longipalpis. In order to evaluate natural infection by Leishmania chagasi and the infectivity of these parasites in the tick, 39 specimens (6 females, 11 males and 22 nymphs) of R. sanguineus were removed from 21 dogs showing diverse symptoms of zoonotic visceral leishmaniasis (ZVL). Six ticks (15.4%) gave positive results for the genus Leishmania using the PCR technique. To determine the infectivity of the parasites, 36 hamsters were inoculated orally and peritoneally with macerates of ticks removed from nine dogs symptomatic for visceral leishmaniasis. After 6 months the hamsters were sacrificed and necropsied. Serum was removed for IFAT, as well as spleen and liver fragments to make imprint smears and for PCR. Eight (88.9%) of these dogs presented ticks that were infective for 14 hamsters (41.2%), 12 (85.7%) of them infected peritoneally and two (14.3%) orally. PCR revealed 27 smears (40.9%) to be positive, 20 (62.5%) of them infected peritoneally and seven (20.6%) orally. IFAT showed 14 positive animals (41.2%). Based on these findings, we suggest that the vectorial capacity of R. sanguineus for L. chagasi should be evaluated further, opening new perspectives in the epidemiology of ZVL.