ContentsOvarian follicles are not homogeneously distributed within the ovarian cortex in several species of mammals. Yet to maximize the reproducibility of experimental results of ovarian transplantation, it is essential to assess the degree of density and distribution of follicles in ovarian tissues before their transplantation. In this study, the ovarian cortex from 13 immature bitches (ten purebred and three mongrels) was sectioned into 1.0-to 1.5-mm 3 cubes, those were fixed, sectioned and stained with haematoxylin and eosin. To evaluate the density and distribution of follicles, the mean number of all stages of follicles per square millimetre was calculated after observation under a microscope. The distribution of follicles was considered even when the variance value was lower than 10 or between 10 and 16, with an absolute value of distortion inferior to 1. The mean number of follicles ranged from 3.24 to 28.34/mm 2 in 25 ovaries from 13 bitches examined. The variance and distortion ranged from 0.35 to 119.64 and −1.87 to 4.40, respectively. The distribution of follicles within the ovarian cortex was judged uneven in 12 of 25 ovaries. These results indicated that follicles were not homogeneously distributed within the ovarian cortex in a large proportion of ovaries.In addition, cryopreserved ovarian fragments with even distribution of follicles were transplanted to NSG mice with or without 400 U/kg of disialylated erythropoietin (asialo EPO). After removing both sides of ovary, a piece of ovarian fragment was placed under the kidney capsule in both sides of kidney. At 4 weeks after transplantation, the fragments were recovered from the mice and the number of primordial, primary, secondary and antral follicles was counted. Total number of follicles and survival rates of follicles in transplanted fragments with asialo EPO were higher than without asialo EPO in four bitches examined. These findings suggest that asialo EPO might be effective on the follicular survival of canine ovarian tissues after xenotransplantation.Knowing the degree of density and distribution of follicles in ovarian tissues before transplantation is expected to contribute to the precise interpretation of results after transplantation of the ovarian tissues.
The cryopreservation of canine spermatozoa facilitates the exchange of genetic material and could be used to improve management programmes for breeding working dogs, such as guide dogs for the blind. When canine spermatozoa are frozen, 'egg yolk' (EY) is added to the diluted semen as a membrane stabilizer and cryoprotectant. We previously reported that, with regard to the cryopreservation of canine spermatozoa, skim milk (SM) is an effective alternative to the conventional cryoprotectant EY, the use of which prohibits the transport of frozen semen during avian influenza epidemics (Abe et al., 2008). In a retrospective study, a single transcervical insemination (TCI) using canine spermatozoa cryopreserved with an SM-based extender resulted in a relatively high delivery rate (83%), whereas an EY-based extender required two TCIs to achieve a comparably high rate (71%) (Abe et al., 2018). Methods for the cryopreservation of spermatozoa have been established in many species. However, long-term equilibration
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