Scanning Electron Microscopic Studies on the Auricular and Atrio-ventricular Valvular Architecture of Pre-natal Non-descript Sheep

The circulatory system plays a vital role in smooth functioning of the body of the cattle Janqueira and Carneiro, 2005) and Uttara fowl (Jaiswal et al., 2017a, Jaiswal et al., 2017b). Heart is the central organ of circulatory system that pumps blood into the blood vessels and performs many vital functions (Sathapathy et al., 2013 and Sathapathy et al., 2014). The faulty development of heart may result in ectopia cordis, dextrocardia, hypoplasia, etc (Sahu et al.,  2021a; Sahu et al., 2021b). Very often, these developmental anomalies of the heart cause foetal death and thereby severe economic loss to the farmers (Sahu et al., 2021). Due to close similarities in many of the systems between the animals and human being, the animals have always become a choice of interest for research purpose, which indirectly help the human being. The detailed ultrastructural study of the auricular and valvular architecture of heart especially in pre-natal sheep has not yet been reported. Hence, the present ultrastructural study was undertaken to elucidate the age wise development of auricles, bicuspid and tricuspid valves of heart in prenatal non-descript sheep.

The foeti of either sex of non-descript sheep were collected from the local slaughter houses situated at Laxmisagar and Jadupur of Bhubaneswar city during the period from July, 2020 to March, 2021 for the present study as a part of the doctoral research work. The adhering amniotic fluid from the body of the foeti was wiped by wet cotton. The crown rump length (CRL) for each foetus was measured in centimetres (cm) with the help of non-stretchable nylon thread and graduated scale. Further, the CRL was placed on the standard CRL-Gestation Age Curve to estimate the approximate age of the foeti in days (Noden and Lahunta, 1985). The collected sheep foeti were divided into three age groups viz. early prenatal (up to 50 days), mid prenatal (51-100 days) and late prenatal (101 to 150 days) The samples of auricles were processed for scanning electron microscopic study and subsequently, the samples were viewed and the photographs were taken in the facility available at Central Instrumentation Facility (CIF), OUAT, Bhubaneswar. The measurements of various parameters of auricles, bicuspid and tricuspid valves of the hearts were also taken at the ultrastructural level by the inbuilt software programming system. The recorded data were subjected to routine statistical analysis (Snedecor and Cochran, 1994).

The present SEM study showed that the myocardium and endocardium were the middle and innermost layers in the wall of the auricles of heart of non-descript sheep in prenatal stage. The present findings were in agreement with the reports of Leeson et al., (1985) in cattle, Eurell and Frappier (2006) in cattle, Dyce et al., (2009) in dog, Widmaier et al., (2011) in human, Konig et al., (2013) in domestic animals, Galfiova et al., (2017) in human and Varga et al., (2017) in human. The endothelial surface of the auricles was occupied by the simple squamous epithelium (Fig 1). The endothelial cells were elongated in shape in all the age groups. There were presence of pores among the endothelial cells in the auricles which were similar to the findings of Sizer et al., (2020) reported in the auricles of heart of Saanen goat.



The subendothelial layer was present in between the inner endocardium and middle myocardium. The present finding was in line with the reports of Myklebust et al., (1975) in sheep, Galfiova et al., (2017) in human and Sizer et al., (2020) in Saanen goat. It consisted of connective tissue fibers and conducting fibers (Fig 6). Further, the subendothelial layer continued with myocardium, the muscle layer of the heart (Fig 6). The present finding was in agreement with the reports of Saunders and Amoroso (2010) in human, Gauvin et al., (2013) in pig and Jaiswal et al., (2017b) in Uttara fowl. It was observed that each muscle bundle in the myocardium consisted of muscle fibers extending parallel to each other longitudinally, and that these fibers made collateral connections with each other in some regions. The present finding was in agreement with the report of Jaiswal et al., (2017b) in Uttara fowl. The pectinate muscles covered the inner side of the right and left atria and were interconnected having a network like appearance (Fig 5). But they are arranged in linear fashion especially in mid prenatal stage, i.e. at 96 days of gestation (Fig 4). There were presence of pores among the pectinate muscles in both the atria in all the age groups under study (Fig 2 and  Fig 3). The present finding was in agreement with the report of Sizer et al., (2020) in Saanen goat. The average diameters of the pores were found to be 172.75±26.12µ, 282.25±52.12µ and 305±13.72µ in the early, mid and late prenatal stages respectively in non-descript sheep at different magnifications. 

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The endothelial surface of the mitral valve was lined by simple squamous epithelium that projected into the lumen of the valve (Fig 7 and Fig 8). The present findings were in line with the reports of Hurle et al., (1985) in human, Icardo et al., (1993) in mouse, Dohmen et al., (2003) in juvenile sheep, Brazile et al., (2015) in pig, Markby et al., (2017) in dog and Sizer et al., (2020) in Saanen goat. Further, the average diameters of the endothelial cells were found to be 3.39±0.24µ and 8.60±0.74µ in the early and mid prenatal stages respectively in non-descript sheep at different magnifications.  

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There was presence of pores among the endothelial cells of the bicuspid valve (Fig 9) in all the age groups under study. The average diameter of the pores was found to be 1.60±0.16µ in the late prenatal stage of the non-descript sheep.

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The subendothelial layer was present just below the endothelium and comprised of connective tissue fibers and conducting fibers (Fig 6). The present observations were in accordance with the findings of Ghonimi et al., (2015) in camel and Sizer et al., (2020) in Saanen goat. The fibers were interwoven with each other forming a network (Fig. 10). The width of the fibers was not uniform and was further categorized into thick and thin fibers especially in the mid prenatal stage, i.e. at 96 days of gestation (Fig 10). The average thickness of thick and thin fibers was measured as 397.5±59.5nm and 119±20.81nm respectively at 96 days of gestation.

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The tricuspid valve guards the right atrio-ventricular orifice, i.e. the opening between the right atrium and right ventricle. The endothelial surface of the valves was lined by simple squamous endothelium that projected into the lumen of the valve (Fig 11 and Fig 13). The present findings were in line with the reports of Hurle et al., (1985) in human, Icardo et al., (1993) in mouse, Dohmen et al., (2003) in juvenile sheep, Brazile et al., (2015) in pig, Markby et al., (2017) in dog and Sizer et al., (2020) in Saanen goat. Further, the average diameters of the endothelial cells were found to be 6.23±0.40µ and 4.86±0.24µ in the early and mid prenatal stages respectively in non-descript sheep at different magnifications. The average longitudinal and transverse diameters of the endothelial cells were recorded as 5.82±0.32µ and 3.41±0.10µ respectively in the late prenatal stage of the non-descript sheep at different magnifications. 

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There was presence of pores among the endothelial cells of the tricuspid valve (Fig 11 and Fig 13) in all the age groups under study. The present finding was in agreement with the report of Sizer et al., (2020) in Saanen goat. The average diameters of the pores were found to be 650±56.29nm and 2.27±0.37µ in the early and mid prenatal stages respectively in non-descript sheep at different magni-fications. There was presence of clusters of pores among the endothelial cells of the tricuspid valve in the heart especially in the late prenatal stage, i.e. at 120 days of gestation period (Fig 14). Further, in late prenatal stage, a lot of variations were recorded in the size of the pores present on the endothelial surface of the tricuspid valve. They were categorized into smaller, medium and larger pores. The average diameters of the smaller, medium and larger pores located among the endothelial cells of the tricuspid valve were noted as 513.83±31.16nm, 1.61±0.07 µ and 3.06±0.15µ respectively in the 120 days gestational age of the foetus at different magnifications.    

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The subendothelial layer was present just below the endothelium of the tricuspid valve and comprised of connective tissue fibers and conducting fibers (Fig 12). The present finding was in agreement with the report of Sizer et al., (2020) in Saanen goat. The subendothelial fibers were interwoven with each other forming a network (Fig 12). The average thickness of the fibers was found to be 2.90±0.24 µ in the 105 days gestational age of the foetus.

The ultrastructural auricular and valvular architecture showed significant variations among different ages in the pre-natal sheep. Further, the present study provided a detailed baseline data on the age wise ultrastructural development of auricles, bicuspid and tricuspid valves of heart in pre-natal sheep that could help in studying various congenital developmental anomalies in different animals.

The authors are grateful to the Officer In-charge, Central Instrumentation Facility (CIF), OUAT, Bhubaneswar for providing necessary facilities and support for the successful completion of this research work within time.