(286) Effect of Maternal and Post-Weaning Supplementation with Zinc Oxide Nanoparticles on Histomorphometry, Stereology, and Oxidative Stress in Ovaries of Santa Inês Lambs

Carolina R. Jimenez ^1* ; Lívia M. Presuto ¹ ; Bruno S. Gatti ¹ ; Willinton P. Pinchao ¹ ; Patrícia S. Corrêa ¹ ; Alessandro FT Amarante ² ; Mário Binelli ³ ; Hélder Louvandini ¹

¹ Centro de energia nuclear na agricultura, Laboratório de Nutrição Animal, Universidade de São Paulo, SP, Brasil.

² Departamento de Biodiversidade e Bioestatística, Instituto de Biociências, Universidade Estadual Paulista Júlio de Mesquita Filho, SP, Brasil .

³ Departamento de Ciências Animais, Universidade da Flórida, Gainesville, FL, Estados Unidos.

Abstract Text:

Zinc is an essential micronutrient involved in various cellular processes, including DNA synthesis, cell division, antioxidant defense, and intracellular signaling. In the ovaries, it plays a role in modulating pathways related to oxidative stress, promoting tissue viability and follicular growth. Zinc oxide nanoparticles (ZnO-np), due to their nanoscale size, have a greater capacity for cellular penetration and may potentiate the cellular effects mediated by zinc. Accordingly, this study aimed to evaluate the effects of maternal and post-weaning supplementation with ZnO-np on follicular dynamics and oxidative stress in ovaries of lambs. Fifty-nine Santa Inês ewes, with a single pregnancy and an average weight of 47.2 ± 1.16 kg, were allocated into three experimental groups at 90 days of gestation: control (placebo with ground hay), ZnO-np (40 nm), and zinc oxide microparticles (ZnO-m) with 300 mg/animal/day until 60 days postpartum (weaning). The ewes were supplemented with 70% ground corn, 15% soybean meal, and 15% cottonseed meal, Aruana grass (Panicum maximum cv. Aruana), Tifton hay (Cynodon spp.), zinc-free mineral salt and water ad libitum. The female lambs (n = 29) remained with their mothers until weaning and subsequently continued under the same treatments until 8 months of age. Their diet consisted of ground corn, soybean meal, Tifton hay (Cynodon spp.), zinc-free mineral salt, and ad libitum water. At 8 months, the ovaries were collected for histological evaluation (follicular classification, tissue normality, morphometry, and stereology). Simultaneously, the production of reactive oxygen species (ROS) was assessed using the dye 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) by confocal microscopy, with quantification of the relative fluorescence intensity performed using ImageJ software. The data were analyzed by ANOVA, followed by Tukey's test for mean comparison (SAS, version 9.4). The results did not show differences between treatments to normality, morphometry, and follicular classification (P > 0.05), except for secondary pre-antral follicles, where the ZnO-np treatment was superior to both ZnO-m and placebo treatments (P < 0.05). Furthermore, the ZnO-np treatment resulted in a higher ovarian follicular population (129,530 cells) compared to the ZnO-m (95,530 cells) and placebo (73,550 cells) treatments (P < 0.05). In mammals, the follicular population is predetermined at birth, with no postnatal renewal. Thus, the number of follicles observed at 8 months (reference value: 80,000 to 120,000 cells) directly reflects the depletion of this stock throughout development. These results indicate that ZnO-np acts as a preservative of the follicular stock, minimizing the accelerated depletion of follicles over time without compromising morphology or tissue integrity. This evidence highlights the potential of nanotechnology in strategies aimed at preserving fertility in animal models and in assisted reproduction contexts.

Keywords: ovário, ROS, população folicular, ovinos.

Financial Support: The authors acknowledge the financial support from the Brazilian National Council for Scientific and Technological Development (CNPq, grant 403541/2021-2) and the São Paulo Research Foundation (FAPESP, grants 2023/13656-3 and 2019/26042-8).