# Type of Extender and Equilibration Time as Factors Affecting Post-Thaw Quality Characteristics of Turkey Semen

## TL;DR

This study shows that turkey semen quality after freezing depends on the extender used and how long it is equilibrated before freezing.

## Contribution

The study identifies optimal extender-equilibration time combinations for improving turkey semen cryopreservation outcomes.

## Key findings

- 20 min equilibration improved motility, membrane integrity, and reduced DNA fragmentation and ROS.
- Beltsville extender provided the best overall sperm quality when used with 20 min equilibration.
- SMM performed best at 40 min equilibration, offering a time-specific alternative for turkey semen preservation.

## Abstract

Avian spermatozoa are highly susceptible to ice-crystal injury, making semen cryopreservation particularly challenging. Post-thaw quality is strongly influenced by the cooling program, equilibration time, extender composition, and choice of cryoprotectants, antioxidants, and antibiotics. Ongoing work therefore seeks species- and even breed-specific optimization of protocols and extenders. In this study, we tested multiple extenders paired with two equilibration times to assess their effects on post-thaw turkey semen quality, revealing that extender performance varied and was dependent on equilibration time.

Commercial turkey breeding relies almost entirely on artificial insemination, yet avian sperm are unusually vulnerable to cooling and freezing injury. As a result, extender chemistry and processing steps, especially low-temperature equilibration, are pivotal for post-thaw performance. We evaluated how extender choice, paired with equilibration time, shapes the post-thaw quality of turkey semen. Ejaculates were diluted in Beltsville, Sperm Motility Medium (SMM), Botucrio, or Kobidil+, then equilibrated for 20 or 40 min before freezing; samples were cryostored for one month and assessed immediately after thawing. The outcomes included motility/kinematics, membrane integrity, mitochondrial activity and membrane potential, apoptosis/necrosis, reactive oxygen species (ROS), DNA fragmentation, and bacteriological load. Overall, 20 min equilibration improved post-thaw motility and membrane integrity, and reduced DNA fragmentation and ROS. Among extenders, Beltsville delivered the best overall sperm quality. Considering the extender × time interaction, Beltsville, Botucrio, and Kobidil+ performed best at 20 min, whereas SMM performed best at 40 min. Thus, Beltsville and SMM provide strong, time-specific options for turkey semen cryopreservation—Beltsville at 20 min and SMM at 40 min.