# Validating Sperm Concentration in Rabbit Cryopreservation Protocol: Implications for Fertility, Litter Size, and Offspring Growth

## TL;DR

This study finds that using specific sperm concentrations in frozen rabbit semen can achieve good fertility and offspring growth, similar to fresh semen, especially when adjusted for the mother's reproductive history.

## Contribution

The study identifies optimal sperm concentrations for cryopreserved rabbit semen based on female parity, improving artificial insemination efficiency.

## Key findings

- Sperm concentrations of 15–35 million per straw achieved fertility rates similar to fresh semen in rabbit does.
- Multiparous does showed higher reproductive performance and heavier offspring compared to nulliparous does.
- Optimal weaning rates were observed across all sperm concentration treatments.

## Abstract

Optimizing reproductive technologies is essential for sustainable rabbit farming and genetic conservation. Cryopreserved semen offers practical advantages, but its use is limited by variable fertility outcomes, partly due to unpredictable sperm concentrations in insemination doses. This study assessed how different concentrations of frozen–thawed sperm affect reproductive performance and offspring growth in nulliparous and multiparous rabbit does. The results demonstrated that low to intermediate concentrations (15–35 million) produced fertility and kindling rates similar to fresh semen, with multiparous females generally achieving higher reproductive performance and heavier offspring. Kit survival and growth were not negatively affected by the use of frozen semen, and optimal weaning rates were observed across all treatments. These findings highlight the importance of adjusting sperm concentration in cryopreservation protocols according to the physiological status of the female, offering practical guidance for enhancing artificial insemination efficiency in rabbit breeding systems.

The cryopreservation of rabbit semen is a valuable strategy for genetic resource preservation and efficient artificial insemination, but outcomes remain inconsistent, partly due to variations in sperm concentration per dose. This study aimed to evaluate the in vivo effects of different sperm concentrations (15, 25, 35, 55, and 75 million per straw) on fertility, prolificacy, and offspring growth in nulliparous and multiparous does. A total of 384 rabbit females were inseminated using frozen–thawed semen, and their reproductive performance was compared with fresh semen. Fertility and kindling rates varied with sperm concentration and parity: nulliparous does showed the highest fertility at 15 million sperm/straw (84.4%), while multiparous does reached peak values at 25–55 million/straw (78.1–81.3%). Litter size and live-born kits were consistently higher in multiparous than in nulliparous does. Offspring body weight at 19 and 60 days was influenced by both sperm concentration and maternal parity, with better growth generally observed in multiparous groups. Weaning success remained high across all groups. Our results indicate that sperm concentrations ranging from 15 to 35 × 106/straw are the most suitable for cryopreservation, as they maintain high fertility, prolificacy, and offspring growth, comparable to fresh semen. These results confirm that optimizing sperm concentration during cryopreservation improves reproductive efficiency and that tailoring insemination strategies to the physiological status of the female enhances outcomes. The results provide useful recommendations for improving cryopreservation techniques in rabbit breeding programs.