Effects of adding different concentrations of myo-inositol on sperm cryopreservation in stallions

• Grant number: 24/18977-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: July 01, 2025
• End date: December 31, 2025
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Eneiva Carla Carvalho Celeghini
• Grantee: Lívia Maria da Silva Paiva
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Cryopreservation of equine semen, despite having several benefits, has some limitations. Therefore, the choice of the diluent to be used in the technique is crucial to protect the sperm during the process. In this sense, myo-inositol has been studied as a component that improves sperm quality, bringing metabolic and morphological benefits to the sperm. However, there are still gaps in fully understanding the potential effect of myo-inositol. Therefore, the objectives of this study are to evaluate the effects of myo-inositol in the cryopreservation of equine sperm, seeking to find an ideal concentration that provides the best benefits on sperm quality. For the following experiment, four stallions will be used, which will undergo an andrological evaluation and analysis of their reproductive history. Four ejaculates will be collected from each stallion (n=16) using a Botucatu model artificial vagina. The semen will be diluted (1:2) in skim milk-based medium and centrifuged. Afterwards, the supernatant will be discarded and the sediment will be resuspended with cryopreservation extender (Botu-Crio®). The concentration will be evaluated and divided into four treatments: CON (control): extender only, MIO15: extender added with 15 mM myo-inositol, MIO30: extender added with 30 mM myo-inositol, MIO45: extender added with 45 mM myo-inositol. The dilution will be performed to a concentration of 200x106 sperm/mL. Subsequently, it will be packaged in 0.5 mL straws and subjected to cryopreservation using an automated freezing system, and the straws will be stored in cryogenic cylinders. Afterwards, two straws from each stallion, batch and treatment will be thawed, homogenized and evaluated for: motility (CASA-iSperm), morphology (using the humid chamber technique), integrity of the plasma and acrosomal membranes, and mitochondrial membrane potential (using fluorescent probes). Statistical analysis will be performed using the Statistical Analysis Systems program (SAS version 9.4; SAS Institute, Inc., Cary, NC, USA). The data obtained will be subjected to analysis of variance (Bartlett's test), evaluated by ANOVA, and the means of the groups will be compared using the Tukey test. A probability of p ¿0.05 will indicate a significant difference. (AU)