Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology

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Effect of lemon essential oil (Citrus limon) and its major components (Limonene (R)-(+) and Limonene (S)-(-)) on cryopreservation of bull spermatozoid

Marlon Castelo Branco , Yndyra Carvalho , Felipe Moraes Junior , Felipe Pereira da Silva Barçante , Felipe Nunes Barros , Luiz Harliton Mota , Geraldo Cortês Carvalho , Ana Abreu Silva , José Souza


Production of reactive oxygen species (ROS) above critical levels affects sperm cell functional integrity and causes oxidative stress. This study was carried to assess the effects of different concentrations (0 µM, 50 µM, 100 µM and 150 µM) of natural antioxidants based on lemon essential oil (Citrus limon) and its major components (Limonene R-(+) and Limonene S-(-)) in supplementing bull semen freezing extender. Cryopreserved sperm cells were submitted to post-thawing computer assisted sperm motility analysis (CASA) to assess sperm kinetics characteristics. ROS were detected by flow cytometry using the association of carboxy-H2DCFDA/propidium iodide-PI stains and epifluorescence microscopy to determine the acrosome integrity of the plasma membrane and mitochondrial membrane potential. Phase contrast microscopy was used to assess vigour and total sperm motility following the heat resistance test (HRT) at times 0, 60, 120 and 180 min. Results demonstrated a better maintenance of the total post-thawing motility of the sperm cells incubated at 37°C for 60 min, when cryopreserved with lemon essential oil at 100 and 150 µM concentrations; no effect was observed on plasma membrane integrity, acrosome integrity and mitochondrial membrane potential when cryopreserved with the addition of lemon essential oil R-(+) and Limonene S-(-). In addition, all the lemon essential oil concentrations were shown to be effective in reducing sperm cell intra-cellular ROS in bulls. In summary, the addition of lemon essential oil (100 and 150 µM) to the cryopreservation medium improved total sperm motility and protected sperm cells against oxidative stress damage.

RD16438  Accepted 13 June 2017

© CSIRO 2017