Histological and Histomorphometrical Characterization of the Cardiac Architecture in Pre-natal Non-descript Sheep (Ovis aries)

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., 2017). 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., 2021a and Sahu et al., 2021b). 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 (Sahu et al., 2021c and Sahu et al., 2021d). The detailed histological and histomorphometrical studies of the cardiac architecture especially in pre-natal sheep has not yet been reported. Hence, the present histological and histomorphometrical studies were undertaken to elucidate the age wise development of cardiac architecture 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. 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) with four animals in each age group. The heart samples were fixed in 10% buffered neutral formalin for 72 hours. After fixation, the tissue samples were processed by Paraffin technique (Bancroft and Stevens, 1964) to obtain 6µm thick paraffin sections. The cut sections were put in the water bath maintained at 37°C and then taken on a clean grease free glass slide. The slides with cut tissue sections on them were air dried and subjected to the routine Haematoxylin and Eosin staining technique for studying the normal architecture, Masson’s trichrome staining technique for presence of collagen fibres, Verhoeff’s stain for presence of elastic fibres and Gomori’s stain for presence of reticular fibres (Bancroft and Stevens, 1964). The H and E stained slides were used for recording the histomorphometrical characteristics after following the standard method of calibration (Culling, 1974). Further, the recorded data were subjected to routine statistical analysis (Snedecor and Cochran, 1994).

The histomorphological observations on the heart showed significant variations among the different age of sheep foeti under study. 

The endothelium of the different chambers of heart, septa and valves had a simple squamous epithelium at all the stages of gestation under the present investigation. The epithelial cells were flat and most of them did not project into the cavity of the heart in the 33 days and 38 days of gestation (Fig 1). Similar features were also observed during 43 days to 96 days of gestation. Some of the epithelial cells appeared to project into the cavity of the heart by the age of 105 days and this feature was maintained during 120 days to 149 days of gestation. These nuclei are reported to act as sensors of shear to detect the strength and direction of blood flow (Tkachenko et al., 2013). The nucleus of the cells was flat and small in the 33 days and 38 days of gestation. Similar features were also observed in 43 days and 49 days of gestation. The nucleus became oval in shape in 59 days of gestation and this character was observed during 72 days to 96 days of gestation. The nucleus was seen to be elongated in outline at age of 105 days and this feature was maintained up to 149 days of gestation. The cytoplasm revealed a strong eosinophilia at 59 days of gestation and this feature was maintained from 72 days till 149 days of gestation.



The subendothelium was very thin and consisted of loose connective tissue at 33 days and 38 days of gestation. Similar features were observed at 43 days and 49 days of gestation. This thin endothelium discerned a dense irregular connective tissue at 59 days of gestation and the feature was found in foeti up to 96 days of gestation (Fig 2). Appreciable amount of smooth muscle fibres, capillaries, arterioles and venules were observed at age of 59 days and the same was also noted during 72 days to 149 days of gestation. Abundance of collagen fibres and moderate quantity of elastic and reticular fibres were observed at age of 105 days of gestation and onwards till 149 days of gestation. The present findings were in agreement with the reports given by Nagpal (1977) in goat and Gupta et al., (2014) in buffalo. The increasingly occurrence of connective tissue fibres particularly the interlacing collagen bundles stranded with smooth muscles with advancing age, clearly advocates for the execution of a dampening effect to the excessive stretch of the cardiac chambers during pumping. In this way, the subendothelium is also reported to protect the cardiac valves from collapse (Banks, 1993). The dense irregular connective tissue bed of the subendocardium became thick at age of 105 days of gestation and the same structural organization was seen from 120 days to 149 days of gestation. It contained scanty smooth muscle fibres and capillaries at 33 days and 38 days of gestation. Similar features were seen at 43 days and 49 days of gestation. The smooth muscle fibres, capillaries, arterioles and venules were observed clearly at age of 59 days and onwards till 149 days of gestation.

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The myocardium formed the bulk of the cardiac wall of the foeti of all the stages of gestation. It was made of cardiac muscle cells or fibres being separated by connective tissue elements in all the ages of gestation. The cardiac muscle cells were cylindrical and branched at all the stages of gestation. The branching pattern of the myocardiocytes was indistinct from the age of 33 days up to 49 days of gestation. The branching pattern was distinctly evident by the age of 59 days and onwards till 149 days of age. The centrally located nucleus of the cardiac muscle cells was oval in shape and was heterochromatic in nature during 33 days to 49 days of age. The nucleus became elongated or ovoid in shape by 59 days of age and retained this shape until 149 days of gestation (Fig 3). The peri-nuclear region of the myocardiocytes was homogenous at all stages of gestation. The cytoplasm of the cells was homogenous and weakly eosinophilic by the age of 33 days and continued with this feature until 49 days of gestation. Similar features were registered at 43 days and 49 days of gestation. The present findings were in agreement with the reports of Sathyamoorthy et al., (2008) in pig foetus and Gupta et al., (2014) in buffalo foetus. The homogenous cytoplasm became moderately eosinophilic between 59 days and 96 days of gestation and strongly eosinophilic by the age of 105 days and onwards till 149 days of gestation. The intercalated discs were seen at the junction between the adjacent cardiac muscle fibres in all stages of gestation in end to end fashion. The discs appeared in broken lines by 33 days of age and continued as such up to 49 days of gestation. They became continuous lines and appeared to be consolidated at 59 days of gestation and maintained this structure from 72 days of age up to 149 days of gestation (Fig 4). The present findings were in agreement with the reports given by Melax and Leeson (1969) in rat and Zhang et al., (1996) in human, but this was in contradiction with the report of Sommer and Johnson (1968) who opined that these discs appeared laterally and longitudinally along the embryonic myocardiocytes.

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Some of the myocardial cells of the right atrium near the opening of the caudal vena cava were large in size and the surrounding myocardiocytes were small in size. These large cells were densely aggregated in the myocardium of the right atrium in the shape of oval patches and linear cords at all the stages of gestation. The oval patches constituted the sinoatrial node and the linear cords formed the internodal fibres. The sinoatrial node and internodal fibres shared similar histomorphological characteristics at all the stages of gestation. The sinoatrial node revealed two types of cells. Most of the cells of the sinoatrial node were intensely stained and consisted of granular cytoplasm with indistinct nucleus (Type-I cells) and the other cells of sinoatrial node had a glassy cytoplasm with a centrally located nucleus (Type-II cells). The cells of the internodal fibres comprised of a glassy cytoplasm with centrally located nucleus at all the stages of gestation.

The epicardium consisted of a simple squamous epithelium or mesothelium supported by a sub-epicardial connective tissue layer at all stages of gestation. The epithelial cells were flat and mostly indistinct at 33 days of age and this feature continued up to 49 days of gestation. They became distinct from the age of 59 days up to 149 days of gestation. The nucleus of the cells appeared flat from the age of 33 days to 49 days of gestation. It became oval at the age of 59 days and this shape was maintained from 72 days to 96 days of gestation. The nucleus became elongated by the age of 105 days and this shape was noted from 120 days to 149 days of gestation. The cytoplasm of the cells was weakly eosinophilic during 33 days to 49 days of gestation. The cytoplasm appeared strongly eosinophilic at 59 days of age and this feature was encountered from 72 days up to 149 days of gestation.

The sub-epicardial connective tissue layer was very thin and consisted of loose connective tissue at 33 days of gestation and onwards till 49 days of gestation. The connective tissue elements became dense irregular type between 59 days and 96 days of gestation. This layer got thick at the age of 105 days and this feature was seen up to 149 days of gestation. The smooth muscle fibres and capillaries were scanty from the age of 33 days to 49 days of gestation. Appreciable amount of smooth muscle fibres, capillaries, arterioles and venules were observed at age of 59 days and similar histological features were also noted during 72 days to 149 days of gestation. The collagen fibres were abundant and both the elastic and reticular fibres were moderate in quantity in the sub-epicardial connective tissue layer at the age of 105 days of gestation which was also maintained during 120 days to 149 days of age of the foetus (Fig 5). A moderate quantity of reticular fibres was noted around the blood vessels at 149 days of gestation (Fig 6). The present findings were in line with the observations of Gupta et al., (2014) in buffalo foetus.

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The histomormetrical observations on the heart showed significant variations among the different age of sheep foeti under study. The average thickness of epicardium, myocardium and endocardium in different parts of the heart were depicted in Table 1. Further, the average diameter of myocardiocytes and their nuclei in different parts of the heart were recorded in Table 2 and Table 3 respectively. The frequency distribution of myocardiocytes, mesenchymal cells and fibroblasts of the myocardium of heart were also showed in Table 4. The recorded histomorphometrical results could not be discussed due to unavailability of the data and research in this field.

The cardiac histology and histomorphometry showed significant variations among different ages in the pre-natal sheep. Further, the present study provided a detailed baseline data on the age wise histological and histomorphometrical development of different cardiac structures in pre-natal sheep that could help in studying various congenital developmental anomalies in different animals.

This is a part of the Doctoral thesis submitted by the First Author to the Odisha University of Agriculture and Technology, Bhubaneswar. The authors are grateful to the Dean, CVSc. and A.H., OUAT, Bhubaneswar for providing necessary facilities and support for the successful completion of this research work within time.

There is no conflict of interests among authors.