Toxicological effect of ethanol on the development of chicken embryo in ova

Toxicological effect of ethanol on the development of chicken embryo in ova. Pure and Applied Biology. Abstract This study aimed at finding out the toxicological effects of ethanol on chicken embryo during early stages of development. The nature and extent of toxicological effects were investigated to identify the concentration which causes the death of the embryo. The total (n=32) eggs were divided into four groups viz. control group, group A, group B and group C. Eight eggs were placed in each group for incubation. Different concentrations of ethanol were given to those groups viz. 5%, 10%, and 15% respectively. Gross anatomical defects particularly embryo’s body weight, body length, wings length and hind limbs length throughout seven days incubation were measured after the exposure of mentioned ethanol concentrations. The results of mean values of seven-day-old chick embryos exposed to 5% ethanol showed non-significant effect ( P >0.05) on body weight, wings length and the hind limbs length of all groups concerning control group chick embryo. But ethanol with a concentration of 10% and 15% showed a significant effect ( P <0.05) on the body length of chick embryo in comparison with 5% treated chicks. The results of hatchability showed that group A showed 90% normal hatchability at the 21 st day while group B showed no remarkable delay in comparison of group C which showed delayed hatching at 22 nd day due to exposure of heavy dose. Moreover, among the most frequent abnormalities, hind limb lengths at 15% and body weight at 5% were found reduced in size. Ethanol exposure increases the risks of developmental defects with increasing embryonic age, delayed and assisted hatching.


Introduction
Alcohol, despite being toxic to humans, is used in industry and home for the manufacturing of compounds like gasoline additives, paints, and perfumes. In general usage, alcohol refers almost always to ethanol, also known as grain alcohol. Ethanol is a colorless, inflammable compound, which is the component of alcohol containing beverages like wine, spirits, and champagne. Ethanol is also used as a solvent in medicines, perfumes, and vegetable essences, such as vanilla. It is also used as a topical antiseptic. Alcohol intake during pregnancy can lead to fetal abnormalities. Prenatal alcohol exposure exerts teratogenic effects on the developing fetus, mainly manifested by growth retardation, abnormal facial features, and Central Nervous System (CNS) damage, collectively known as the Fetal Alcohol Syndrome (FAS). Alcohol also causes cardiovascular anomalies [1]. Consumption of alcohol during pregnancy consequences of fetal alcohol syndrome (FAS), which include unprompted abortion, growth impedance, facial anomalies, and mental retardation. Alcohol produces its effects by narrowing of placental and umbilical cord blood vessels, decreasing the oxygen supply to the fetus. Placental measurements noticed from normal births demonstrated that umbilical blood vessels squeezed in the presence of alcohol even at concentrations as low as 0.05%, the amount checked in 1 to 1.5 drinks. Oxygen deficiency may play a role in the development of FAS-associated with mental retardation and behavioral deficiencies [2]. Ethanol exposure accelerates the cells died within the area of cranial neural crest during embryogenesis, has been revealed to result in loss of the SHH gene (Sonic hedgehog) as well as transcripts involved in SHH signaling pathways. The SHH gene is responsible for the production of the Sonic Hedgehog protein which is an important morpho gene that shows a role in cell growth, cell specialization and the prefiguring of the body [3]. Thus, the CNS seems to be very susceptible to developmental exposure to alcohol across all stages of development with gross physical defects consequential from exposure at peri-organogenetic periods and insufficient in distinct neuronal populations when exposure takes place at the postnatal period or the end of gestation. Recently, with the introduction of new tools, variations in gene expression patterns associated with the above-stated neurulation or gastrulation phase, ethanol sensitive openings have initiated to develop new insight on the molecular mechanisms that involved in ethanol teratogenicity [4]. An essential issue in the research studies of developmental effects of alcohol is that the comparative roles of alcohol itself and that of its primary metabolite, acetaldehyde. The second is known to have teratogenic effects [5]. Furthermore, the maternal metabolic illness role of alcohol with or without placental dysfunction is undefined in the pathogenesis of FAS [6]. According to Gilbert, 2003, there is marked the difference in the capability of mothers and fetuses to metabolize alcohol, and it is supposed that 30-40% of the children will have FAS, born to mothers who drink alcohol during pregnancy [7]. Some of these complications can be overwhelmed by using the chick embryo, which does not metabolize alcohol to acetaldehyde until day 9 of gestation [8] and therefore permits the study of ethanol effects in the absence of its primary metabolite. The chick embryo is an attractive model to study developmental procedures. It has been used from so long as a replacement of human embryos due to their close biological similarity. In the early periods, chick embryo morphology is very analogous to human [9]. The study was aimed to investigate the effects of in ovo ethanol exposure with different concentrations on early developmental stages. The nature and magnitude of these effects were recorded to detect the concentration lethality which causes gross structural abnormalities along with their hatchability characteristics.

Materials and methods
Chick embryos were exposed to different ethanol levels and compared with same age controls. The study was carried out in the Laboratory of Zoology, The Department of Life Sciences, The Islamia University Bahawalpur, Bahawalpur Pakistan by the approval of the ethical committee.

Handling of eggs
A total of 32 fertilized, non-incubated, White Leghorn eggs (each weighing about 60g) were collected from Govt. poultry farm Bahawalpur, Punjab, Pakistan. All eggs were divided into four groups Viz. Control, A, B and C having eight (n=8) individuals in each. Four individuals from every group were sacrificed at day 7 th of development and compared with the same aged individuals of the control group. The remaining was kept constantly incubated till the day of hatching. The eggs were candled to pinpoint air cavity, and the unfertilized eggs were excluded from the experiments. Eggs were cleaned by 70% ethanol for aseptic measurements. To check the alcoholic teratogenicity in the chick, fertilized eggs were marked, numbered and divided into four groups. Including control, there were placed eight (n=8) eggs in each group. A hole of diameter 2mm in the center of air sac was made by egg driller in all eggs of all groups. The concentration of 5%, 10% and 15% of ethanol was injected into the eggs of group A, group B, and group C respectively. The hole was sealed by using melted paraffin. All the eggs were left at room temperature in an upright position for 2 hours. After that, all were shifted to the incubator for incubation at 37⁰C with 55% relative humidity. Eggs which were cracked or stored in the refrigerator were excluded from the study. The total incubation period of chicken was 21 or 22 days.

Examination of embryos
Four embryos from every group at 7 th day were dissected and observed the effects of different concentrations of alcohol on total body weight, body length, wings length, and hind limbs length by cutting chorioallantoic membrane and amnion. Hatchability characteristics of the remaining four chicks from each respective group were also observed. Most of the chicks hatched at day 22 whereas the remaining chicks that could not hatch by natural way till day 22 were taken out manually by flouting the shells. The newly hatched chicks were inspected for gross anomalies, abnormal posture, gait and other abnormal behavior as compared to the control.

Statistical analysis
All ethanol-exposed the group's means and standard errors values regarding embryo's body weight, body length, wings length, and hind limbs length were calculated. The Tuckey's Multiple Comparative Test was used for post-hoc analysis. Percentage of newly hatched chicks with gross abnormalities and reduction of all alcoholexposed groups concerning particular developmental defects was examined.

Results
A total of 32 eggs were placed under experimental care those were divided into four (4) groups viz group A, group B, group C, and control group. There were different concentrations of ethanol 00, 5%, 10%, and 15% were injected in that groups of eggs correspondingly to note its effect on body weight, body length, wings length and size of hind limbs length of chicks embryo. Measurements regarding body weight, body length, wings length, and hind limbs length were calculated at seven (7th) day of incubation while the other characteristics were noted on hatching at 21st or 22nd day.

Effect of ethanol on body weight
The results of weight according to the effect of different concentrations of ethanol are given in (Table 1 & Figure 1). Its findings revealed that all the concentrations of ethanol showed no significant effect on the weight of chicks concerning the means values of control group individuals.       (Table  4 & Figure 4). According to that results the concentrations of ethanol showed no significant effect on hind limbs length of Chick embryos of all groups concerning the comparison with control group Chicks embryos.  The most frequent abnormalities The most frequent abnormalities among body weight, body length, wings length and hind limbs length of the entire group embryo were calculated concerning the control group. The body weight of the group with 5% ethanol exposure was found reduced while at 15 % the hind limbs length of all the groups concerning control showed a high rate of abnormality. There was no significant effect on body length and wing length abnormalities regarding all groups (Table 5).  (Table 6) ( Figure 5).
. Table 6. Day of hatching among ethanol-exposed groups

Mode of hatching
There were few of the embryos produced normal at the time of hatchability, and few were abnormal. All embryos of the control group and group A were normal, two embryo of group B and one embryo of group C were also normal. But two embryo of group B and three embryo of group C were produced abnormal which are given in (Table 7 & Figure 6).

Discussion
In this study ethanol effects of different concentrations, i.e. 00%, 5%, 19%, 15% on chicken embryo early developmental stages, their characteristics viz body weight, body length, wings length, and hind limbs length were observed. Eggs were divided into four groups, i.e. group Control, group A, group B and group C concerning ethanol concentration respectively. There was no significant (P>0.05) ethanol effect on weight, length of wings and hind limbs of the chick embryo of all groups vs. control group. Lower no of the abnormal embryo was found in all groups compared to the control group. Ethanol effects the chick embryo body length significantly (P<0.05) in group B and group C with concentration 19% and 15% respectively. It was in line with the findings reported that ethanol effect significantly on the heart chambers and become a cause of FAS supports the finding of this study [16,17]. FAS reported by Kiecker and Boronat are in line with this study [18]. They described ethanol cause a significant effect on craniofacial development in chick embryo during their early stage. Measurements were taken for embryo body length, wing bud length and leg bud length over a course of twenty one (21) days on a sample of 12-hours embryos that were exposed to four selected ethanol concentrations; concentration first is control in which no injection of ethanol, concentration second is (5%), concentration third is (10%) and concentration fourth is (15%) [3]. The developmental and general growth effects as well as effects on skeletal muscles were studied by exposing the embryos chicks to single doses of 5%, 10%, and 15% ethanol. There a significant hindrance was found in head circumference, crown-rump length, and body weight in ethanol-exposed chicks. These parameters were compared with results of control groups, and it was noticed that this growth retardation was linked with a substantial and proportionate decline in the weight of skeletal muscles [19]. In this research, 56% of chicks presented reduced mobility and were inept at standing upright. Additional possible cause of hypo-activity could be alcohol removal. The researchers exposed male Sprague Dawley rats to ethanol vapors for 12 or 14 days and then examined their locomotion activity. Hypo-activity appeared promptly in rats during ethanol removal. Extraction induced hypo-activity was noticed in previous studies directed on various species of rats after ethanol intake through various routes [20,21]. In the present study, the experimental group C exhibited a major failure of yolk sac withdrawal into the abdominal cavity. Generally, yolk sac starts withdrawing into the abdominal cavity at 19th day and this withdrawal is finished at day 20 [22]. There a substantial growth delay was found in these chicks, which is in agreement with our study. In a previous study, chick embryos were grown in shell-less cultures with supplementation of a single dose of 50% ethanol. Ethanol considerably enhanced the mortality rate and encouraged the growth impedance [23]. Experimental studies on various species revealed that oxidative stress, apoptosis, distorted cell cycle, blocked DNA and protein synthesis are some of the significant causes of ethanol persuaded malformations [24,25]. No notable anomalies were found in live and dead chick embryos of group A and B in the experimental group, which demonstrated that both increase period of exposure and enhanced embryonic age increases the risk of developmental defects and growth hindrance in developing chick embryos.

Conclusion
In this study we found that the ethanol exposure enhanced the risks of developmental fetus defects with increased embryonic age, detained hatching and more assisted hatchings. Newly hatched chicks, which were exposed to alcohol exhibited reduced locomotion activity and poor balance which may be observed to damage skeletal muscle and central nervous system in growing embryos. These outcomes might have significance concerning the possible developmental effects of alcohol misuse at various stages of human pregnancy and the devising proper advice and involvement policies.