Sperm traits on in vitro production (IVP) of bovine embryos: Too much of anything is good for nothing

RESEARCH ARTICLE Sperm traits on in vitro production (IVP) of bovine embryos: Too much of anything is good for nothing Adriano Felipe Perez Siqueira1, Letı́cia Signori de Castro1, Patrı́cia Monken de Assis1, Luana de Cássia Bicudo1, Camilla Mota Mendes1,2, Marcı́lio Nichi3, José Antônio Visintin2, Mayra Elena Ortiz D’Ávila Assumpção1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Laboratory of Spermatozoa Biology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil, 2 Laboratory of in vitro Fertilization, Cloning and Animal Transgenesis, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil, 3 Laboratory of Andrology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil * Abstract OPEN ACCESS Citation: Siqueira AFP, de Castro LS, de Assis PM, Bicudo LdC, Mendes CM, Nichi M, et al. (2018) Sperm traits on in vitro production (IVP) of bovine embryos: Too much of anything is good for nothing. PLoS ONE 13(7): e0200273. https://doi. org/10.1371/journal.pone.0200273 Editor: Peter J. Hansen, University of Florida, UNITED STATES Received: January 9, 2018 Accepted: June 23, 2018 Published: July 10, 2018 Copyright: © 2018 Siqueira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Grant number: 2013/07940-9 (AFPS), Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES); (AFPS), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Grant number: 2016/15147-5 (MEODAA). The funders had no role in study design, data collection and analysis, Sperm samples used on fertilization strongly influence the in vitro production (IVP) rates. However, sperm traits behind this effect are not stated consistently until now. This study aimed to evaluate the isolated and combined effect of some sperm traits (MB: total motility before Percoll® gradient, MA: total motility after Percoll® gradient, AI: acrosome integrity, MI: membrane integrity, MP: mitochondrial membrane potential, and CR: chromatin resistance) on IVP rates. This is the first study focusing on the isolated effect of distinct traits. For this purpose, the experiment was divided in three steps. In first step, to study behavior of traits sperm samples (n = 63 batches) were analyzed and ranked based on each trait. In second step, samples ranked were selected from target ranks regions and allocated in groups of four to five batches, creating Higher and Lower groups, according to two different approaches. One aimed to form groups that differed to all sperm traits simultaneously (effect of combined traits). The other aimed to form groups that differed only to a single sperm trait while no differences were observed for the remaining traits (effect of each isolated trait). In third step, for each group successfully formed in step 2, sperm samples were individually and prospectively used for IVP. Cleavage, embryo development and blastocyst rates were recorded and compared between Higher and Lower of respective trait groups. Surprisingly, evaluation of isolated effects revealed that lower levels of MB, AI and MP resulted in higher embryo development and blastocyst rates (p<0.05), which was not observed on cleavage rate. We conclude that sperm traits strongly influence embryo development after in vitro fertilization (IVF), affecting the zygote competence to achieve blastocyst stage. Individually, levels of MB, AI or MP could be some of the key traits that may define IVP efficiency on current systems of embryo production. PLOS ONE | https://doi.org/10.1371/journal.pone.0200273 July 10, 2018 1 / 16 Sperm traits on IVP: Too much of anything is good for nothing decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction In vitro production (IVP) of bovine embryos has allowed the use of a waste biological resource from valuable females to increase their offspring in breeding programs, particularly in Brazil [1,2]. Nevertheless, more than half of IVP embryos fail to reach the blastocyst stage during in vitro culture (IVC). Intuitively and mistakenly, this suggests that IVC step is the main responsible for the IVP failures [1]. However, Rizos et al. [3] showed that during IVC, in vivo matured oocytes achieved higher blastocyst rate than in vitro oocytes (58.2% versus 38,9%) and in vivo fertilized oocytes result in higher blastocyst rate compared to in vitro fertilized ones (73.9% versus 58.2%), regardless the similar cleavage rates. Such results may indicate that problems during in vitro maturation (IVM) and in vitro fertilization (IVF) steps could potentially impair the blastocyst production mainly during embryo development rather than first cleavages. In addition to IVF shortcomings, the bull effect has been generally related as a cause of variation on IVP rates, including differences between batches from same bull [4–18]. In this context, attempts to select sperm samples with improved in vitro performance based on sperm features would be an interesting tool to indicate, beforehand, IVP performance and improve embryo production yields [9]. However, to predict the fertilization ability of a given sperm sample by sperm traits analysis is still a long way to achieve any substantial results using the current approaches. This is probably due to the unknown importance of sperm traits analyzed and the disregards of possible confounding effects such as bull effect and interactions among traits. In attempt to better understand the relation between sperm traits and IVP rates, we chose sperm traits previously suggested as possible predictor candidates for IVP performance: motility [9,14,16,17,19], status of acrosome [7–9,13,14,16,19], plasma membrane [9,13,14,16,19], chromatin [16,19–21] and mitochondria [9,21], and we evaluated their effect on IVP. These sperm traits play fundamental roles during fertilization process and embryo development and together imply the status of the main function and cellular structures of spermatozoa. The present study was designed to identify which sperm traits are important to determinate IVP yields. With this information, it will be possible to select samples that could provide higher IVP yield to guide the improvement of sperm production and handling used to IVP, and also to elaborate strategies to upgrade IVF step. While these traits have been previously studied, some confounding effects (...truncated)