_____________________________________________________________________________________________________ *Corresponding author: E-mail: ofemora66@unam.mx, ofemora2001@yahoo.com.mx; Asian Research Journal of Agriculture 15(4): 36-44, 2022; Article no.ARJA.88166 ISSN: 2456-561X In-vitro Evaluation of Three Conservation Methods of Probiotics from 42-day-old Tropical Calves Sarahí Rodríguez-González a , Laura González-Dávalos a , Armando Shimada a and Ofelia Mora-Izaguirre a* a Laboratorio de Rumiología y Metabolismo Nutricional, Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, Blvd. Juriquilla 3001, Juriquilla 76230, Querétaro, México. Authors’ contributions This work was carried out in collaboration among all authors. Author SRG manages the analyses of the study, designed the study, performed the statistical analysis, wrote the first draft of the manuscript, and managed the literature searches. Author LGD helped with the experiments and reviewed the manuscript. Author AS reviewed the manuscript. Author OMI designed the study, reviewed the manuscript. All authors read and approved the final manuscript. Article Information DOI: 10.9734/ARJA/2022/v15i430165 Open Peer Review History: This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://www.sdiarticle5.com/review-history/88166 Received 06 April 2022 Accepted 13 June 2022 Published 16 June 2022 ABSTRACT Aims: The conservation of probiotics is of great importance due to the microorganism’s viability; therefore, their properties and benefits depend on it. In this work, it was studied three methods of conservation; refrigeration, cryopreservation, and lyophilization of two probiotics isolated from the rumen of 42 day-old calves, previously evaluated: Animal 1-day 42 (A1D42) and Animal 3-day 42 A3D42. Place and Duration of Study: Laboratorio de Rumiología y Metabolismo Nutricional, Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, between August 2020 and September 2021. Methodology: It was evaluated the viability of the probiotics after of 0, 30, 60 and 90 days of refrigeration, cryopreservation, and lyophilization as preservation methods. The effect of the lyophilized probiotic A1D42 on the digestibility in vitro, such as its effect on the volatile fatty acids (VFAs) production was also determined. Original Research Article