Influence of environmental temperature on the cooling curve and viability of equine spermatozoa

• Grant number: 24/13144-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: January 01, 2025
• End date: December 31, 2025
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Gabriel Augusto Monteiro
• Grantee: Marina Lucena Fredou
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

Semen refrigeration is a widely used biotechnology in the equine industry. This technique maximizes the use of stallion´s semen, prevents the spread of sexual diseases, eliminates geographical barriers and, most importantly, increases the longevity of the semen. The preservation of cooled sêmen is achieved through controlled temperature reduction (cooling curve) and consequent reduction of sperm metabolism. However, variations in the environmental temperature can significantly influence the cooling curve and impact semen quality. Therefore, the aim of this experiment is to evaluate the influence of three temperatures (5°C, 22°C, and 37°C) and four extenders on the cooling curve and sêmen quality. For this study, six ejaculates from six healthy stallions, aged between 8 and 12 years, in a routine collection schedule, will be used. Prior to the study, the stallions will undergo a complete andrological examination. Ejaculates will be collected using an artificial vagina and will be fractionated for subsequent dilution until reaching a concentration of 50 x 106 spermatozoa/mL in the extenders: BotuSêmen®, BotuTurbo®, BotuSêmenSpecial®, and BotuGold®. After initial processing, samples will be stored in cooling boxes with six different cooling curves: 1) 5°C curve with 1 ice pack; 2) 22°C curve with 1 ice pack; 3) 37°C curve with 1 ice pack; 4) 5°C curve with 2 ice packs; 5) 22°C curve with 2 ice packs; and 6) 37°C curve with 2 ice packs. Semen samples will be evaluated at the following times: fresh (T0), cooled for 8 hours (T8), and 24 hours (T24) regarding sperm kinetics and plasma membrane integrity. Sperm kinetics analysis will be performed using a computer-aided sêmen analysis system (Hamilton Thorne Research - IVOS 12, Beverly, MA, USA), which measures the total sperm motility, progressive sperm motility, track speed, linear velocity, curvilinear velocity, and the percentage of sperm with rapid movement. Additionally, an aliquot will be allocated for plasma membrane integrity analysis using epifluorescence microscopy. Data will be analyzed with ANOVA using repeated measures to compare the effects of treatments on sperm kinetics and plasma membrane integrity over time. Greenhouse-Geisser correction will be applied when necessary to adjust the sphericity of the data and a 5% statistical significance level will be used (P < 0.05).