Maternal stress in sheep during late pregnancy influences sperm quality in early puberty of the offspring F  abio Luís Henrique a , Helena Viel Alves Bezerra a , Heloise Zavatieri Polato a , Arícia Christofaro Fernandes a , Adroaldo Jos  e Zanella b , Maíra Bianchi Rodrigues Alves b , Eneiva Carla Carvalho Celeghini b , Leonardo Batissaco b , Ricardo de Francisco Strefezzi a , Lina Fernanda Pulido-Rodríguez a , Henrique Barbosa Hooper a , Cristiane Gonçalves Titto a, * a Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de S~ ao Paulo, Av. Duque de Caxias Norte, 225, Campus Fernando Costa, Pirassununga, SP, 13635-900, Brazil b Faculdade de Medicina Veterin aria e Zootecnia, Universidade de S~ ao Paulo, Av. Duque de Caxias Norte, 225, Campus Fernando Costa, Pirassununga, SP, 13635-900, Brazil article info Article history: Received 13 December 2018 Received in revised form 27 September 2019 Accepted 9 October 2019 Available online 15 October 2019 Keywords: Developmental programming Lipopolysaccharide Ram Testicular development Ultrasound abstract Stress at the end of sheep gestation can damage the reproductive development of young males. The aim of the present study was to evaluate the effects of LPS administration in the last third of sheep pregnancy on the reproductive parameters of prepubertal rams. Thirty-six pregnant nulliparous ewes (12 ± 2 months old; 45 ± 6 kg) were assigned to two treatments, LPS (E. coli; 0.8 mg kg 1 ) and control (placebo/ saline) administered in late pregnancy (120 days post-conception). The animals gave birth to 17 male lambs (11 LPS; 8 control). Reproductive development of the young rams was analyzed from 5 to 12 months of age. A completely randomized design in double factorial scheme was used. The data were analyzed by analysis of variance. The model included treatment (LPS; control), age as main effects and their interactions, and the animal as a repeated measure. Means were compared by the PDIFF-SAS (Pr > | t|) at P < 0.05. An effect of age was observed for scrotal circumference, testicular consistency, homoge- neity of testicular parenchyma, vascularization, semen quantity and quality, and blood testosterone concentration (P < 0.05). LPS increased sperm defects (P < 0.05) but an interaction with age was not observed (P > 0.05) with higher abnormalities only during months 8 and 9 (P < 0.05) and not thereafter. In summary, LPS did not cause long-term damage to testicular morphology analyzed from the onset of puberty to sexual maturity. However, LPS treatment affected sperm morphology during early puberty of the offspring. © 2019 Elsevier Inc. All rights reserved. 1. Introduction Prenatal stress is an adverse event experienced by the mother during pregnancy [1 ,2] that can influence epigenetic transgenera- tional activity according to the type of trauma, intensity, duration, consecutive exposure, age, and sex [3]. Lipopolysaccharide (LPS) is a molecule found in the outer membrane of gram-negative bacteria that elicits immune responses and is widely used in animal ex- periments to simulate stress or disease. In late pregnancy, LPS can affect the stress axis and alter fetal development programming, causing residual damage throughout the animal’s life [4]. In sheep, similar to laminitis, systemic challenges such as inflammation caused by gram-negative bacteria can act as a major stressor depending on the stage of pregnancy [4]. As the frequency of this infection in sheep flocks is extremely high [5,6], the in- fections caused by these microorganisms can trigger the activation of the immune system, which results in the release of the endotoxin LPS from the outer membrane [7e9]. However, little is known so far about the effects of prenatal stress on the development of the fetal reproductive system [10, 11]. In specific stages of pregnancy, prenatal stress can negatively in- fluence the formation of the neuroendocrine system which, in * Corresponding author. E-mail address: crisgtitto@usp.br (C.G. Titto). Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com https://doi.org/10.1016/j.theriogenology.2019.10.008 0093-691X/© 2019 Elsevier Inc. All rights reserved. Theriogenology 145 (2020) 158e166