Scanning Electron Microscopic Studies on the Ventricular Architecture of Pre-natal Non-descript Sheep

The circulatory system plays a vital role in smooth working of the body of the animal (Jaiswal et al., 2017a, Jaiswal et al., 2017b and Janqueira and Carneiro, 2005). 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. 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 ventricular architecture of heart especially in pre-natal sheep has not yet been reported as evidenced from the available literature. Hence, the present ultrastructural study was undertaken to elucidate the age wise development of ventricles 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). Based on prenatal age, the collected sheep foeti were divided into three age groups viz. early prenatal (1-50 days), mid prenatal (51-100 days) and late prenatal (101 to 150 days). The samples of ventricles were processed for scanning electron microscopic study (Scanning Electron Microscope, Make: Hitachi and Model: S-3400N) 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 ventricles 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).

It was revealed that the endocardium was the inner layer of the ventricles of heart. It covered the inner surface of the heart and consisted of a simple squamous epithelium. The endothelial cells were elongated in shape and arranged linearly on the surface of ventricles in 33 days prenatal non-descript sheep (Fig 1 and Fig 2). Further, the boundaries among the endothelial cells were indistinct in this age group. The endothelial cells of a row appeared cord like. As the age advanced, i.e. during mid prenatal period at about 96 days of gestation, it was noted that the shape and size of the endothelial cells varied significantly (Fig 3). They were elongated to oval in shape with significant variation in their sizes. There was presence of pores among the endothelial cells on the surface of the ventricles. In late prenatal period, the endothelial cells of the ventricles became elongated with pores among them. 



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The average longitudinal diameters of the endothelial cells were found to be 5.00±0.36µ and 17.51±1.48µ in the early and mid prenatal stages respectively in non-descript sheep at different magnifications. Further, the average transverse diameter of the endothelial cell was observed as 2.55±0.09µ in the early prenatal stage of non-descript sheep. There was presence of pores among the endothelial cells on the surface of the ventricles. The average diameters of the pores were found to be 1.03±0.11µ, 2.21±0.27µ and 2.87±0.28µ in the early, mid and late prenatal stages respectively in non-descript sheep at different magnifications. 

The subendocardial layer was present in between the inner endocardium and middle myocardium. It consisted of connective tissue fibers and conducting purkinje fibers (Ghonimi et al., 2015 and Sizer et al., 2020). They were quite predominant in the late prenatal period, i.e. at the age of 120 days of gestation (Fig 4). The endocardial fibers were distinctly coiled around the endothelial cells of the ventricles in this age group (Fig 5).

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The myocardium was the middle layer of the ventricles of heart present between the inner endocardium and outer pericardium (Myklebust et al., 1975 and Galfiva et al., 2017). It consisted of cylindrical and highly branched cardiac muscle fibers (Fig 6). 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 (Jaiswal et al., 2017b). There was presence of pores all over the surface of the myocardium of the ventricles (Fig 7).  The average diameters of the pores were found to be 5.13±0.48µ and 11.31±1.45µ in the mid and late prenatal stages respectively in non-descript sheep at different magnifications. 

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The papillary muscles were ridge like structures projecting into the lumen of the ventricles of heart (Fig 8). Further, there were presence of bands of chordae tendinae that connected the papillary muscles and the bicuspid and tricuspid valves (Fig 8). The sizes of the chordae tendinae varied within the ventricles of the animal. The average length of chordae tendinae was measured as 2.33±0.15mm in the early prenatal stage. Similarly, the average width of chordae tendinae was recorded as 500±20.21µ, 146±6.09µ and 142±7.25µ at the base, middle and apex respectively in the early prenatal stage at different magnifications.  

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It was noted that the endothelial surface of the chordae tendinae consisted of endothelial cells that varied significantly in size among the age groups (Morse et al., 1984). The endothelial cells were oval in shape in early prenatal stage (Fig 9), which gradually became elongated in the mid and late prenatal stages (Fig 10 and 11). The average diameters of the endothelial cells were found to be 3.03±0.17 and 10.05±0.46µ in the early and late prenatal stages respectively in non-descript sheep at different magnifications. The average longitudinal and transverse diameters of the endothelial cells were noted as 5.93±0.45µ and 3.06±0.35µ respectively in the mid prenatal period of the animal at different magnifications.

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It was observed that the pores of various sizes were arranged linearly in patches on the endothelial surface of the chordae tendinae of the ventricles especially in the late prenatal period, i.e. at about 105 days of gestation (Fig 12). The pores were categorized as larger and smaller types. The average diameters of the larger and smaller pores were found to be 3.48±0.46µ and 533.5±62.53nm respectively in the late prenatal stages of the animals under study at different magnifications. 

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The ultrastructural ventricular 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 ventricles of heart in pre-natal sheep that could help in studying various congenital developmental anomalies in different animals.

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