Histochemical and Biochemical Developmental Studies on the Heart in Pre-natal Non-descript Sheep ( Ovis aries )

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INTRODUCTION
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 andSathapathy 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 histochemical and biochemical study of the cardiac architecture especially in pre-natal sheep has not yet been reported.Hence, the present histochemical and biochemical study was undertaken to elucidate the age wise development of cardiac architecture in prenatal nondescript sheep.

MATERIALS AND METHODS
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 Histochemical and Biochemical Developmental Studies on the Heart in Pre-natal Non-descript Sheep (Ovis aries) 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 and processed by routine Paraffin technique (Bancroft and Stevens, 1964) to obtain 6µm thick paraffin sections.The sections were stained by PAS technique for neutral mucopolysaccharides, Alcian blue (pH: 1.0 and 2.5) for acidic mucopolysaccharides, Thionin method for mast cells and the staining for demonstration of argentaffin cells by Singh's modified Masson-Hamperl reaction for endocrine cells.Further, the fresh cut tissue samples were prefixed in formol calcium at 4  C for 24 hours to obtain the frozen tissue sections of 10 m thickness in cryostat for demonstration of acid phosphatase activity by azo-dye coupling technique and for fat by Sudan Black B method (Bancroft and Stevens, 1964).
For estimation of various enzymes like Alkaline Phosphatase, Creatine Kinase, Serum Glutamic-Pyruvic Transaminase, (SGPT), Serum Glutamic-Oxaloacetic Transaminase (SGOT), fresh heart tissue was employed for preparation of tissue extract.For preparation of tissue extract, fresh tissue sample was collected from wall of left and right ventricles of heart.The tissue was then, mixed with 10 ml of PBS and triturated by a tissue homogenizer.The homogenized tissue was centrifuged at 5000 rpm for 10 min and the tissue extract (supernatant) was used for estimation of different cardiac enzymes in a manner similar to techniques used in serum.The enzyme estimation was done using Kits (manufactured by Coral Clinical Systems, Tulip Diagnostics (P) Ltd., Goa, India) as per the standard protocol given by the manufacturer of the Kit itself and the protein estimation was carried out by SDS-PAGE method.The biochemical data obtained from the present investigation were subjected to routine statistical analysis (Snedecor and Cochran, 1994).

RESULTS AND DISCUSSION
The present histochemical and biochemical studies revealed significant results with regards to the micro architecture and enzymatic activity in different structures of the heart in prenatal non-descript sheep.

(i) Histochemical study (a) Right atrium
The endocardium of auricle showed mild PAS activity in all ages of gestation.The PAS activity was fair in the myocardium of auricle in foeti aged 33 days to 49 days of gestation and it became strong during 59 days to 149 days of gestation in sheep.The endocardium of pectinate muscle illustrated mild PAS activity at all ages of gestation.The PAS activity was fair in the myocardium of pectinate muscle in sheep foeti aged 33 days to 49 days of gestation that became strong from 59 days of gestation and onwards till term end.The endocardium of crista terminalis showed mild PAS activity in all ages of gestation.The PAS activity was fair in the myocardium of crista terminalis between the age of 33 days and 49 days of gestation that became strong during 59 days to 149 days of gestation.The endocardium of intervenous crest showed mild PAS activity in all ages of gestation.The PAS activity was fair in the myocardium of intervenous crest in sheep foeti aged 33 days to 49 days of gestation that became strong from 59 days to 149 days of gestation.The neutral mucins are reported to yield a positive reaction and chiefly reflect a carbohydrate component or a glycoprotein in the tissue.The initial low content of the PAS positive material in the early gestation age of the sheep foetus followed by a moderate rise of the material within the myocardium may suggest for maintaining the membrane integrity as well as myocardial activity with increasing age of the sheep foetus.An absence of an alcianophilia suggests that probably the acid mucopolysaccharides and sulphated mucopolysaccharides do not play a role during cardiac development in animals.
Few mast cells were encountered in the interstitial connective tissue of the myocardium of the pectinate muscles in all stages of gestation.Though the exact role of such a sparse mast cell population in unclear, but they are reported to bear a wide variety of chemicals such as histamine, heparin, cytokines and growth factors.They are likely to play a role in the immune status of the cardiac tissue.Appearance of this cell in the heart during prenatal period might reflect acquiring immuno-competency during embryogenesis.Few argentaffin cells with endocrine granules were observed in the myocardium of the pectinate muscles in foeti of 72 days of age (Fig 1).These cells are probably playing an endocrine role to regulate the extracellular fluid volume during embryogenesis of the cardiac tissue.
The nerve fibres were scanty in the endocardium of auricle of heart of sheep foeti from age of 33 days to 49  days of gestation and the fibres became fair in number from 59 days to 149 days of gestation.The nerve fibres were few in the myocardium of auricle in foeti aged 33 days to 49 days of gestation and they were fair in frequency from age of 59 days to 96 days of gestation.They became moderate in amount from 105 days to 149 days of gestation.The nerve fibres did not appear in the endocardium of pectinate muscles between the age of 33 days and 49 days of gestation.They were few in the said location by the age of 59 days till the age of 149 days of gestation.The nerve fibres were few in the myocardium of pectinate muscles during 33 days to 49 days of gestation which became fair in number from the age of 59 days to 96 days and moderate in amount from the age of 105 days till term end.The nerve fibres could not be traced in the endocardium of crista terminalis at 33 days and 38 days of gestation.By 43 days and 49 days of gestation, the fibres were sparse.They were few in the endocardium of the foeti aged 59 days and onwards till 149 days of gestation.The nerve fibres were scanty in the myocardium of crista terminalis between 33 days and 49 days of gestation.They were fair in amount during 59 days to 96 days of gestation and moderately branching fibres were seen in foeti aged 105 days and onwards.The nerve fibres were occasional in the endocardium of intervenous crest in foeti of from age of 33 days to 49 days of gestation.They were scanty in foeti aged 59 days to 96 days of gestation and were few in amount from the age of 105 days to 149 days of gestation.The nerve fibres were scanty in the myocardium of intervenous crest from 33 days of age to 49 days of gestation.They were few in foeti by the age of 59 days to 96 days of gestation and fair in amount from age of 105 days to 149 days of gestation.The increase in neuronal supply is suggestive of an increased neural control over the differentiating and maturing cardiac tissues.
The acid phosphatase activity was feeble in the endocardium of pectinate muscles between 59 days and 149 days of gestation.The myocardium of crista terminalis and intervenous crest showed mild acid phosphatase activity during 33 days to 49 days of gestation and a fair activity was found from 59 days to 149 days of gestation.This enzyme is known to impart a lytic function and therefore, presence of this enzyme in higher concentration at a later age of the foetus within the myocardium may ask for a role in the process of differentiation and cell growth.
A mild grade lipid deposition was seen in the endocardium of auricle during 105 days to 149 days of gestation.The lipid deposition was mild in the myocardium of auricle from the age of 33 days up to 49 days of gestation which became fair from 59 days to 96 days of gestation and a moderate lipid deposition was evident during 105 days to 149 days of gestation.A feeble lipid deposition was seen in the endocardium of pectinate muscles during 59 days to 96 days of gestation and it became mild during 105 days to 149 days of gestation.The lipid deposition was feeble in the myocardium of pectinate muscles from age of 33 days to 49 days of gestation that became mild in foeti aged 59 days to 96 days of gestation.A mild lipid deposition was noted in foeti aged 105 days up to age of 149 days of gestation.The exact nature of this kind of age dependent distribution is not clear though the present findings corroborate with the reports of Sathyamoorthy (2003)  gestation and moderate in amount in foeti of age 105 days to 149 days of gestation.Similarly, fair numbers of nerve fibres were observed in the myocardium of moderator band from the age of 33 days to 49 days of gestation.The nerve fibers were fair in number in the endocardium of papillary muscles in foeti from age of 59 days to 96 days of gestation and moderate in amount in foeti of 105 days, 120 days, 136 days and 149 days of gestation.
The myocardium of chordae tendineae showed moderate acid phosphatase activity in the foeti between 105 days and 149 days of age.Similarly, the acid phosphatase activity was moderate in the myocardium of moderator band, ventricular wall and papillary muscle from 105 days of age to 149 days of gestation.The lipid deposition was fair in the myocardium of papillary muscle in foetus from the age of 105 days to 149 days of gestation (Fig 3).Moderate lipid deposition was revealed in the epicardium during 96 days to 149 days of gestation.The exact nature of this kind of age dependent distribution is not clear though the present findings corroborate with the reports of Sathyamoorthy (2003) in pigs.

(c) Left atrium
The PAS activity was moderate in the endocardium of auricle and myocardium of pectinate muscle during 59 days to 96 days of gestation.Few mast cells were encountered in the myocardium of the pectinate muscle and interatrial septum in all stages of gestation (Fig 4).Few argentaffin cells with endocrine granules were observed in the myocardium of the pectinate muscle in all stages of gestation.The nerve fibres were few in foeti from age of 59 days to age of 96 days and became fair in frequency during 105 days of age to 149 days of gestation in the endocardium of auricle.Similarly, the nerve fibers were moderate in number in the myocardium of auricle, pectinate muscle and interatrial septum in foeti aged 105 days to 149 days of gestation, whereas fair amount of nerve fibers were noted in the endocardium of pectinate muscle at the age of 105 days to 149 days of gestation.
The acid phosphatase activity was moderate in the myocardium of auricle, atrial wall, pectinate muscles and interatrial septum during 59 days to 96 days of gestation, whereas strong activity was noted in 105 days to 149 days of gestation (Fig 5).The epicardium revealed moderate acid phosphatase activity reaction by 59 days to 81 days of age followed by an intense reaction by 96 days of age till term end.The lipid deposition was fair in the myocardium of auricle during 105 days to 149 days of gestation.The myocardium of pectinate muscle, interatrial septum revealed fair amount of lipid deposition in foeti aged 59 days to 96 days of gestation, whereas moderate lipid deposition was observed in foeti aged 105 days to 149 days of gestation.The epicardium revealed moderate deposition of lipid during 96 to 149 days of gestation.The exact nature of this kind of age dependent distribution is not clear though the present findings corroborate with the reports of Sathyamoorthy (2003) in pigs.

(d) Left ventricle
The PAS activity was intense in the myocardium of the ventricle and papillary muscle at the age 149 days of the sheep foeti.The endocardium of interventricular septum showed moderate PAS activity during 59 days to 96 days of gestation, whereas the activity was strong in the myocardium of interventricular septum from the age of 33 days to 49 days of gestation.Few mast cells were encountered in the myocardium of the papillary muscle and interventricular septum in all stages of gestation.Few argentaffin cells with endocrine granules were observed in the myocardium of the papillary muscle in all stages of gestation.
The nerve fibres were fair in quantity in the endocardium of chordae tendineae in foeti between 105 days and 149 days of gestation.The myocardium of chordae tendineae consisted of fair amount of nerve fibres from the age of 59 days to 96 days of gestation and they were moderate in frequency in foeti aged 105 days to 149 days of gestation (Fig 6).The nerve fibres were moderate in frequency in the myocardium of papillary muscle and interventricular septum from 59 days to 96 days of gestation and became abundant from 105 days to 149 days of gestation.
The acid phosphatase activity was moderate in the myocardium of chordae tendineae, papillary muscle in foeti aged 105 days and onwards till 149 days of gestation.The lipid deposition was fair in the endocardium of papillary muscle in foeti aged 105 days to 149 days of gestation.The lipid deposition was moderate in the myocardium of ventricle, papillary muscle and interventricular septum from the age of 105 days and onwards till term end.The epicardium revealed moderate deposition of lipids in sheep foeti during 105 days to 149 days of gestation.The exact nature of this kind of age dependent distribution is not clear though the present findings corroborate with the reports of Sathyamoorthy (2003) in pigs.

(ii) Biochemical study
The creatine kinase activity in right ventricle of heart of the sheep foetus was measured as 752.13±72.31U/l, 2285.24±210.45U/l and 6723.85±662.27U/l in early prenatal, mid prenatal and late prenatal stages respectively.The activity of creatinine kinase in left ventricle of heart of the sheep foetus was found to be 895.21±83.24U/L, 3267.43±310.41U/l and 9730.80±968.21U/l in early prenatal, mid prenatal and late prenatal stages respectively (Fig 7).The increase in creatine kinase activity in both the ventricles with age resulted in higher energy reserves in the myocardium for better pumping efficiency of heart in the late prenatal life of the sheep foeti (Hildegard et al., 1990).The increased in the number of protein bands with age in the sheep foeti might be contributing to the increased interaction of actin-myosin and might be involved in providing structural support or elasticity to the cardiac muscles (Hartzell and Sale, 1985).

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

Fig 1 :
Fig 1: Photomicrograph of right atrium of heart of sheep foetus aged 120 days showing distribution of argentaffin cells in the myocardium (arrow).
Singh's modified argentaffin reaction  400 Histochemical and Biochemical Developmental Studies on the Heart in Pre-natal Non-descript Sheep (Ovis aries)

Fig 2 :
Fig 2: Photomicrograph of right ventricle of heart of sheep foetus aged 33 days showing intense PAS activity in the epicardium (arrow) and a moderate activity in the myocardium (arrow head).

Sudan black B  100 Fig 3 :Fig 4 :Fig 5 :
Fig 3: Photomicrograph of right ventricle of heart of sheep foetus aged 149 days showing distribution of lipids in the epicardium (arrow) and myocardium (arrow head).
The average alkaline phosphatase activity in right ventricle of heart of the sheep foetus was estimated as 10.36±0.13U/l, 19.59±1.73U/l and 51.53±4.73U/l in early prenatal, mid prenatal and late prenatal life respectively.The average alkaline phosphatase activity in left ventricle of heart of the sheep foetus was 10.96±0.93U/l, 20.64±1.63U/l and 53.12±4.71U/l in early prenatal, mid prenatal and late prenatal stages respectively (Fig 8).

Fig 6 :
Fig 6: Photomicrograph of left ventricle of heart of sheep foetus aged 149 days showing distribution of nerve fibres.

Fig 7 :
Fig 7: Photograph showing Creatine kinase (CK) activity in the wall of left ventricle (LV) and right ventricle (RV) of heart of sheep foeti during early, mid and late prenatal period.

Fig 8 :
Fig 8: Photograph showing Alkaline phosphatase (ALP) activity in the wall of left ventricle (LV) and right ventricle (RV) of heart of sheep foeti during early, mid and late prenatal period.

Fig 10 :
Fig 10: Photograph showing Serum glutamic oxaloacetic transaminase (SGOT) activity in the wall of left ventricle (LV) and right ventricle (RV) of heart of sheep foeti during early, mid and late prenatal period.

Fig 9 :
Fig 9: Photograph showing Serum glutamic pyruvic transaminase (SGPT) activity in the wall of left ventricle (LV) and right ventricle (RV) of heart of sheep foeti during early, mid and late prenatal period.

Fig 11 :
Fig 11: Photograph showing calibration curve for determination of molecular weight of protein bands obtained from tissue extract from the of wall left ventricle (LV) and right ventricle (RV) of heart of sheep foeti during early, mid and late prenatal period.

Fig 12 :Fig 13 :
Fig 12: Gel electrophoresis of the ventricular tissue extract of heart of sheep foetus showing the protein bands of different molecular weights at the age of 43 days, 49 days and 81 days of gestation.