Optimization of spermatozoa analysis in mice: A comprehensive protocol.

Sperm quality is critical to predict reproductive alterations caused by immunological factors or toxicant agents. Yet, no detailed protocol has been published focusing on analyses of sperm parameters in mice. Our aim was to evaluate the most efficient diluent for mice sperm analyses and to optimize the sperm morphology classification, through the comparison of different staining methods. The diluents assessed were PBS (baseline), HTF, DMEM, 1 % BSA in PBS and 9 % skimmed powdered milk diluted in PBS. Spermatozoa were evaluated for vitality, motility, and morphology, smears were stained with Papanicolaou, HE, Giemsa, and Rapid staining. Sperm vitality and total motility reached better scores in milk based and DMEM diluents. HE raised up as an effective option since its combination with any of the diluents we tested, resulted in a fair staining, which was appropriated to evaluate mice spermatozoa. Finally, based on WHO manual, we have updated the current morphological classification for mice sperm, since we have detailed the head defects as well as included midpiece and tail defects on it. Taken together, we presented a useful, low cost, and reliable method to assess sperm morphology that could be employed worldwide by laboratories dedicated to study reproductive biology on mice model.

Sperm production and seminal analyses in a Neotropical sperm-storing vespertilionid bat yellowish myotis (Myotis levis).

Yellowish myotis is a Neotropical vespertilionid bat that presents a seasonal reproduction. The sperm is produced in the Mature stage, stored in the Regressed stage and released in the Rest stage (mating period). Aiming to understand, for the first time, the relationship between testis and epididymis physiology in yellowish myotis reproduction, the spermatogenesis length, sperm production, and seminal parameters were herein evaluated. Fifty-one adult male bats were captured in Santuário do Caraça, Minas Gerais, Brazil. The gonads were collected in the Maturing and Mature stages for histomorphometric and immunohistochemical analyses, whereas the epididymis was evaluated in all reproductive stages for seminal studies. Our results demonstrated that the yellowish myotis spermatogenic process is fast, lasting 31.70 ± 0.15 days. Despite the low Sertoli cell efficiency (6.60 ± 1.23), the high numbers of Sertoli cells per testis enable an elevated sperm production in the Mature stage. The sperm concentration, vitality, and motility presented the highest values in the Regressed stage; however, in this period, an increased incidence of sperm morphological defects was detected. In the following period (Rest stage), a drastic reduction of defective sperm was observed, suggesting quality control of sperm before the mating period. Furthermore, the epididymis ability to maintain a long-term sperm-storage was observed in 26.7% of the bats in the Maturing stage. In summary, yellowish myotis presented a fast and high sperm production during the Mature stage. These sperms are stored and selected before mating period.