Revista Cienfica, FCV-LUZ / Vol. XXXV Recibido: 26/01/2025 Aceptado: 06/10/2025 Publicado: 28/10/2025 hps://doi.org/10.52973/rcfcv-e35650 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 6 Revista Cienfica, FCV-LUZ / Vol. XXXV hps://doi.org/10.52973/rcfcv-e35730628 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico The impact of ethanol exposure during fetal development on the rena of chicken embryos El impacto de la exposición al etanol durante el desarrollo fetal en la rena de embriones de pollo 1,3 University of El Oued, Faculty of Life and Natural Sciences, Department of Agronomy. El Oued 39000, Algeria ²University of Batna 2, Faculty of Natural and Life Sciences, Department of Ecology and Environment, 05000 Batna, 53 Route de Constanne. Fesdis, Batna 05078, Algeria ⁴University of Mohamed Khider, Faculty of Nature and Life and Earth Sciences and the Univers,Department of Veterinary Sciences, 07000 Biskra, Algeria ⁵University of Batna 2, Faculty of Natural and Life Sciences, Department of Biology of Living Organisms.Batna (05110), Algeria ⁶Mohamed CherrifMessaadia University, Instute of Agronomic and Veterinary Sciences, Laboratory of Science and Technic of living. Souk Ahras, Algeria ⁷Universityof Batna1, Instute of Veterinary Science and Agricultural Sciences, Department of Veterinary Sciences, ESPA Laboratory.Batna 05000, Algeria *Corresponding author: boussouar-hadjira@univ-eloued.dz ABSTRACT It is common knowledge that excessive consumpon of alcohol by pregnant women may lead to neonatal mortality, a delay in fetal development and serious malformaons. In this study, we used the chicken-embryo model to demonstrate the ethanol teratogenic effect on the development of the rena. Forty-nine ferlized eggs were employed in our experiment. As a result, the chicken embryos were injected with varying amounts of ethanol in the air space (10%, 30%, and 50%). The eggs were kept in a humid incubator with a temperature of 37 °C and a humidity level of 60–65%. On the 20 th day of incubaon, the eggs were cracked and the chicks were examined with their length and body weight taken into account. These operaons were followed by the resecon of the ocular globes for a histological examinaon. The eyes were fixed with formalin at 10%. The right eye was seconed at 5μm with a Leica microtome. Hematoxylin-Eosin staining was, then, applied on the slides, which were also observed under a microscope. The opc-microscopic observaon of the ocular samples of each group has revealed that the in ovo ethanol exposure has caused rena changes, including the internal plexiform and the ganglion-cell layers. In treated groups the thickness of these two layers decreased in comparison with the control group. In conclusion, the current findings indicated that treang chicken embryos with ethanol at an early stage resulted in significant abnormalies and a lower embryo survival rate. Ethanol has harmful effects on the embryonic development including growth retardaon and ocular abnormalies; this effect is dose-dependent. Key words: Alcohol; chicken embryo; malformaon; rena; teratogenic effect RESUMEN Es bien sabido que el consumo excesivo de alcohol por parte de las mujeres embarazadas puede provocar mortalidad neonatal, retraso en el desarrollo fetal y malformaciones graves. En este estudio, ulizamos el modelo de embrión de pollo para demostrar el efecto teratogénico del etanol sobre el desarrollo de la rena. En esta invesgación experimental se ulizaron 49 huevos fecundados. Para ello, se inyectaron diferentes dosis de etanol en los embriones de pollo al (10%, 30% y 50%) en el espacio aéreo. Los huevos se incubaron en una incubadora húmeda a una temperatura de 37 °C y a un 60- 65% de humedad. Al vigésimo día de incubación, se cascaron los huevos y se examinaron los pollitos teniendo en cuenta su longitud y su peso corporal. A estas operaciones siguió la resección de los globos oculares para un examen histológico. Los ojos se fijaron con formol al 10%. El ojo derecho se seccionó a 5μm con un micrótomo Leica. A connuación, se aplicó la nción de hematoxilina-eosina en los porta objetos, que también se observaron al microscopio. La observación óptico-microscópica de las muestras oculares de cada grupo ha revelado que la exposición in ovo al etanol ha provocado cambios en la rena, incluidas las capas plexiforme interna y ganglionar. El grosor de estas dos capas disminuye en comparación con el grupo de control. En conclusión, los hallazgos actuales indican que el tratamiento de embriones de pollo con etanol en una fase temprana provoca anomalías significavas y una menor tasa de supervivencia embrionaria. El etanol ene efectos nocivos en el desarrollo embrionario, como retraso del crecimiento y anomalías oculares; este efecto depende de la dosis. Palabras clave: Alcohol; embrión de pollo; malformación; rena; efecto teratogénico Hadjira Boussouar* 1 , Fahima Neffar 2 , Aicha Mouane 3 , Sara Benrezak 4 , Ramzi Lamraoui 5 , Tarek Khenenou 6 , Omar Bennoune 7 3 Department of Biology of Living Organisms, Faculty of Natural and Life Sciences, University of Batna 2, Batna (05110), Algeria

The impact of ethanol exposure during fetal development on the rena of chicken embryos / Boussouar et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Among the various teratogenic agents, alcohol is considered the most prevalent worldwide. Maternal alcohol consumpon during pregnancy has been widely documented as a major risk factor for developmental abnormalies, notably leading to fetal alcohol syndrome (FAS) and fetal alcohol spectrum disorders (FASD). Given the significance of this issue, any statement regarding the prevalence or impact of teratogens should be supported by quantave data, and ideally complemented by comparave analyses highlighng their principal causes [1]. Given the growing number of women who drink alcohol globally [2], this is a very significant situaon. Despite the fact that the rate of alcohol use has been largely stable over the past 30 years, pregnant women’s alcohol consumpon increased from 12.4% in 1991 to 16.3% in 1995. FAS cases also doubled in number between 1979 and 1993, with an incidence rate of 6.7% per 10.000 live births in 1993 [3]. An elevated infant mortality, head and facial malformaons, a microcephaly, and organ malfuncon are just a few of the potenally harmful clinical effects of alcohol use during pregnancy [4 ,[5]. Renal degeneraon and opc nerve hypoplasia are the most oſten seen defects in children with FAS. Alcohol disrupts the development of the rena, resulng in a delayed and faulty cell migraon as well as a loss of neurons in the interior layers of the rena, especially the ganglion cell layer. The number of myelin-axons in the layer of opc nerve fibers has been significantly decreased [6]. Researchers have ulized a variety of animal models to invesgate the causes and consequences of various alcohol- related disorders. Such models have aided researchers in their exploraon of the ways that both short- and long-term alcohol intakes can obstruct the normal development of embryos [7]. An incredibly helpful model in developmental biology, embryology, and teratology is the chicken embryo (Gallus gallus domescus). There are many sources that provide an accurate and comprehensive descripon on the growth of chicks [8].The use of chicken models has a number of benefits for developmental study. Ferlized eggs are readily available, affordable, and don’t need an incubator to grow. To directly view or manipulate the embryo, a hole can be simply made in the eggshell. The eggshell can potenally be simply destroyed to connue developing [9]. Because there is no in ovosystem in the maternal metabolism, teratogenic effects cannot be directly isolated. Briefly stated, the chicken-embryo model provides a realisc and affordable model in which modern experimental tools may be used to understand how a teratogen affects parcular pathways underpinning organogenesis and morphogenesis [8]. The chicken eye has become a preferred animal model in experimental ophthalmology because of its quick growth, superior visual abilies, and ease of general manipulaon. It is frequently employed in the research of parasympathec funcons in the development and ageing [10]. The purpose of this study is to examine the teratogenic effects of in ovo ethanol exposure on the renal development in a system of chicken embryos, taking into account the injected ethanol dose and the stage of embryonic development. MATERIAL AND METHODS Ethical approval Throughout the trial, the instuonal animal ethics commiee and naonal animal welfare requirements were adhered to accordingly. Guidelines of the Local Ethical Commiee. The study is registered under the reference 03/2024. Experimental design The experimental study was done on a set of 49 embryonated eggs of the broiler chickens (Hubbard F15) strain collected at the private hatchery of Agricultural Cooperave Errakhaà WaIzddihar, located in Theniyetsaida (Ain Yagout – Batna, Algeria). The eggs were divided into five groups: untreated group, group treated by 0.9% NaCI and three groups exposed to ethanol at different concentraons (10%, 30% and 50%) (TABLE I). The ferlized chicken eggs of each group were injected once with100µl of ethanol. TABLE I Treatments and number of eggs in each group of study Group Treatment Number of eggs Untreated group No-treated 7 NaCl-treated NaCl (0.9%) 9 Treated; ethanol EtOH (10%) 10 EtOH (30%) 11 EtOH (50%) 12 NaCl: Sodium Chloride, EtOH: Ethanol. Injecon of substances This parcular process is essenal and demands the highest level of sterility. In order to inject the chemical into the air sacs using a graduated syringe in microlitre, a Bunsen burner (OMM, Italy), equipped with a tap and a flame stabilizer, was used [11 , 12 ,[13]. Incubaon The eggs were automacally turned every four hours (h) during the 480 h (20-day) incubaon period in a humid incubator (Real 49 automac, Italy) set at 37 °C and 60–65% humidity [6 , 11]. Overall length and body weight measurement of the chicks On the 20 th day of incubaon, the eggs were cracked and the embryos were examined.Their lengths were measured using a caliper (RS PRO, China), and their body weights were determined 2 of 6

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico with a balance (KERN, Germany). To idenfy the overall length and body weight of the chicks, the extra-embryonic membranes were removed and the samples were placed on absorbent paper during 2 to 3 min. Each measure was repeated four mes. Histological examinaon The samples (eyes) were fixed in buffered formalin at 10% passed through a series of ethanol showers from 80 to 100%, then cleared with Xylene during one h in each ethanol shower and then incorporated in paraffin Wax (Histosec Paslles) Sigma-Germany bath at 58 °C [14 ,[15]. 5 µm secons of the right eye were taken with a microtome (Leica Jung-histocut 820, Germany), Following hematoxylin and eosin staining (H&E), the slides were examined using a Carl Zeiss Axioskop 20 opcal microscope (Germany) that was aached to a DOM 300 digital video camera [16 , 17 , 18 , 19]. The image was viewed using Oasis soſtware (Oasis Scienfic Inc.) and analyzed using Image J, a powerful image analysis program developed by the Naonal Instute of Health [20]. Stascal analysis of the experimental data The stascal analysis of the experimental data was carried out using Microsoſt Excel 2010 Soſtware and Graph Pad Prisme 5.01. The data concerning the chicks’ viability were analyzed using the chi-square test. The data concerning the chicks’ body weight and length, and the rena layers thickness were analyzed using the Mann- Whitney U test. The data were presented in average ± standard deviation (SD). A difference of P<0.05 between the ethanol- exposed and control groups was deemed stascally significant. RESULTS AND DISCUSSION The effects of ethanol on chicks’ viability The results have revealed a slight difference in the survival rate of chicks treated with 0.9% NaCI (77.8%) and 10% ethanol (70%) compared to the untreated group (85.7%). When the ethanol dose was increased to 30% and then to 50%, the survival rate further reduced to (54.5%) and (50%), respecvely (TABLE II). This indicates that the mortality of 20-day-old chicks varies between groups and that their viability declines in a dose-dependent manner. These data clearly show that ethanol exposure significantly increased early embryo mortality. This finding is consistent with previous studies by Boussouar et al. [21]; Kamran et al. [22]; Shabtai and Fainsod [23]; and Laghari et al. [24], which also reported that early-stage ethanol treatment reduces the survival rate of chicken embryos. TABLE II Average of survival rates of chicks treated with ethanol Groups Treatment Number of eggs Number of living chicks Living chicks (%) Untreated group No-treated 7 6 85.7 NaCl-treated NaCl (0.9%) 9 7 77.8 Treated; ethanol Ethanol (10%) 10 7 70 Ethanol (30%) 11 6 54.5 Ethanol (50%) 12 6 50 NaCl: Sodium Chloride The effects of ethanol on body weight of the chicks Concerning the measurements, the results revealed a significant decrease in the body weight of chicks exposed to high doses of ethanol (30 and 50%), with mean values of (27.77 ± 2.15 and 24.82 ± 2.33g), respecvely. These values were significantly lower (P<0.05) than those recorded in the group treated with 0.9% NaCl, which showed averages of (30.94 ± 2.34 and 30.31 ± 2.69g) (TABLE III). This dose-dependent reducon in body weight indicates that ethanol exposure leads to a marked delay in prenatal growth. These findings are in agreement with Naqi et al. [25], who reported a similar decline in chick body weight following ethanol treatment. Furthermore, they confirm previous observaons by Tufan and Saroglu-Tufan [11], as well as studies by Rao and Chaudhuri [26]; Smith et al. [27]; and Laghari et al. [24], which demonstrated that ethanol exposure even during the pre-hatching stage can negavely affect the health and development of avian embryos. Commonly observed effects include teratogenic effects, reduced hatchability, lower hatch weight, and impaired post-hatch growth. In addion, Munir et al. [28] reported that even low doses of ethanol do not improve poultry producvity and that moderate to high doses may negavely affect welfare and performance traits. TABLE III The body-weight and length averages of the chicks Groups Body weight of the chicks (Average ± SD) Length of the chicks (Average ± SD) Untreated group 30.94 ± 2.34 14.97 ± 0.68 NaCl (0.9%) 30.31 ± 2.69 14.50 ± 1.63 EtOH (10%) 28.27 ± 4.61 13.97 ± 1.43 EtOH (30%) 27.77 ± 2.15* 13.48 ± 0.43* EtOH (50%) 24.82 ± 2.33* 13.37 ± 0.80* NaCl: Sodium Chloride, EtOH: Ethanol, SD: Standard deviaon,*: values in the same column are significantly different (P<0.05). 3 of 6

The impact of ethanol exposure during fetal development on the rena of chicken embryos / Boussouar et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico The effects of ethanol on body length of the chicks Also, a significant decrease in the overall length of the chicks was observed in the ethanol-exposed groups, parcularly those treated with (30 and 50%) ethanol, which showed average lengths of (13.48 ± 0.43 and 13.37 ± 0.80 cm), respecvely. These values were significantly lower (P<0.05) compared to the untreated group. Ethanol thus appears to reduce chick embryo body length in a dose-dependent manner. These findings are in agreement with those reported by Afzal et al. [29]. The effects of ethanol on the rena microscope structure The microscopic examinaon of renal samples from each group using an opcal microscope revealed notable histological changes depending on ethanol concentraon. In the control groups (untreated and 0.9% NaCl-treated), the two renal layers (the internal plexiform layer and the ganglion-cell layer) showed normal organizaon. However, in the group exposed to 10% ethanol, there was a significant reducon (P<0.05) in the thickness of both the internal plexiform layer (54.67 ± 0.69 µm vs. 60.31 ± 0.49 µm in controls) and the ganglion-cell layer (22.11 ± 0.56 µm vs. 28.70 ± 0.32 µm in controls) (FIG. 1). Control 10% EtOH 0 20 40 60 80 Groups Thickness of the inner plexiform layer (µm) Control 10% EtOH 0 10 20 30 40 Groups Thickness of the ganglion layer (µm) FIGURE 1. Effect of exposure to ethanol (10%) on the chick’s rena (Hubbard F15), Algeria. A.Effect of EtOH (10%) on the internal plexiform layer; B. Effect of EtOH (10%) on the ganglion layer. These alteraons became more pronounced in the group exposed to 30% ethanol, which exhibited a marked decrease (P<0.01) in the thickness of the internal plexiform layer (42.89 ± 1.19 µm) and ganglion-cell layer (18.99 ± 0.54 µm) compared to controls (FIG. 2). Control 30% EtOH 0 20 40 60 80 Groups Thickness of the inner plexiform layer (µm) Control 30% EtOH 0 10 20 30 40 Groups Thickness of the ganglion layer (µm) FIGURE 2. Effect of exposure to ethanol (30%) on the chick’s rena (Hubbard F15), Algeria. A. Effect of EtOH (30%) on the internalplexiform layer; B. Effect of EtOH (30%) on the ganglion layer. The most severe changes were observed in the 50% ethanol group, where the internal plexiform layer and ganglion- cell layer showed significant thinning (42.33 ± 0.92 µm and 19.92 ± 0.27 µm, respecvely; P<0.001) relave to controls (FIG. 3). Control 50% EtOH 0 20 40 60 80 Groups Thickness of the inner plexiform layer (µm) B A B A A 4 of 6

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Control 50% EtOH 0 10 20 30 40 Groups Thickness of the ganglion layer (µm) FIGURE 3. Effect of exposure to ethanol (50%) on the chick’s rena (Hubbard F15), Algeria. A. Effectof EtOH (50%) on the internal plexiform layer; B. Effect of EtOH (50%) on the gan- glion layer. Overall, these findings demonstrate that in ovo exposure to ethanol leads to renal degeneraon in a dose-dependent manner, parcularly affecng the internal plexiform and ganglion-cell layers, which supports the observaons reported by Tufan et al. [12]; Guerri and Pascual [30] and Holahan et al. [31]. CONCLUSION This study aimed at clarifying the teratogenic effect of prenatal ethanol exposure at early stages of the development on the rena in a chicken-embryo model. Ethanol has a direct effect on embryonic viability, causing a dose-dependent decrease in embryo survival. Ethanol caused a significant delay in prenatal growth, as shown by dose-dependent measurements of mean body weight and chick length. The chicks exposed to ethanol showed a change in microscopic structure of the rena. This change has been observed at the level of the internal rena layers; in parcular, the internal plexiform and the ganglion- cell layers whose thickness has decreased in comparison to the control group. This decrease is closely correlated to the delivered dose. In conclusion, ethanol negavely impacts embryonic development, major abnormalies in the structure of the rena and growth retardaon. Conflict of interests The authors have no conflict of interest to declare in regard to this publicaon. BIBLIOGRAPHIC REFERENCES [1] Li ZY, Ma ZL, Lu WH, Cheng X, Chen JL, Song XY, Chuai M, Lee KK, Yang X. 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