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Kerala Veterinary and Animal Sciences University, Wayanad
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ThesisItem Open Access HISTOMORPHOLOGY AND ULTRASTRUCTURE OF THYROID AND ADRENAL GLANDS IN LARGE WHITE YORKSHIRE PIGS REARED AT HIGH AND LOW ALTITUDES(COLLEGE OF VETERINARY AND ANIMAL SCIENCES POOKODE, 2022-04-04) SIMANTA PATGIRI; Rajani C.V.Thyroid and adrenal glands of the Large White Yorkshire pigs reared at high and low altitude was studied to investigate the comparative morphology, micrometry and ultrastructure and serum hormone level in animals reared at low and high altitude. Materials for the study were collected from 24 Large White Yorkshire pigs of one year old categorized into four groups with respect to sex and altitude. Each group consisted of 6 animals. The thyroid and adrenal glands were collected from each animal. Observations on morphology, histology, histochemistry, electron microscopy, morphometry and micrometry were carried out and data were analyzed statistically to find out any significant difference. The thyroid gland in Large White Yorkshire pig was laterally covered by the cranial thymus. The position of the gland varied from the caudal border of the larynx to a position dorsal to the manubrium sterni. Thyroid resembled a single centrally-placed gland but after dissection, it revealed two lobes connected by an indistinct isthmus. The average weight of the thyroid gland of both male and female animals in low altitude was more than high altitude. The average length of intact thyroid gland in male and female was more in high altitude compared to low altitude. The average width and thickness of the intact thyroid gland in both male and female groups were more in low altitude than the corresponding high altitude group. The capsule of thyroid gland extended as fine trabeculae of interlobular connective tissue into the parenchyma of the gland and separated it into numerous lobules. The thyroid follicle had a maximum diameter of 301.35 μm. The diameter of thyroid follicles was more in the central area and it reduced towards the peripheral area. Parafollicular or C-cells were present in 3 locations viz. interfollicular, intrafollicular and parafollicular position. The diameter of the thyroid follicle in the high altitude male group was significantly higher than that of the low altitude male group but it was reverse in case of female animal. The thyroid follicle diameter was higher in female compared to male animals irrespective of altitude. The adrenal glands were detected either cranio-medial or entirely on the medial surface of the kidney and embedded entirely within the perirenal fat of the kidney. In low altitude male group, the average weight of right and left adrenal was higher than the corresponding high altitude group but it was reverse in case of low altitude female animals. In the low altitude, the average thickness of right and left adrenal was higher than that of high altitude for both male and female groups. In low altitude female group, the average length of right and left adrenal was lower than that of high altitude female group. In low altitude female group, the average thickness of right and left adrenal was more than that of high altitude group. Histologically, the adrenal gland presented outer cortex and inner medulla. The gland was entirely surrounded by a dense and distinct fibrous connective tissue capsule. The inner zone of the capsule extended very thick trabeculae enclosing blood vessels at right angles from the capsule, at regular intervals into the cortex towards the medulla. However, trabeculae were indistinct from zona fasciculata inwards. A few trabeculae enclosing blood vessel extended even upto the medulla. The boundary between the deeper part of the capsule and zona glomerulosa layer revealed a group of relatively undifferentiated 'capsular' cells. The junction of zona glomerulosa and zona fasciculata presented lightly stained undifferentiated cells were observed. For both male and female animals, the average thickness of zona glomerulosa of right and left adrenal gland were significantly higher in high altitude group than the respective low altitude group. The zona reticularis in the sow was usually wider than that in the boar in all the groups studied. In male pigs, the average serum T3, T4 and cortisol level were significantly higher in low altitude group compared to that of high altitude group. In female pigs, the average serum T4 and cortisol level were significantly higher in low altitude group compared to the high altitude group. The average serum cortisol level in males were significantly higher than that of female animals in both low and high altitude. Irrespective of sex, the average serum T3, T4 and cortisol level were significantly higher in low altitude group than that of high altitude group.ThesisItem Open Access STRUCTURAL AND PHYSICO-CHEMICAL EVALUATION OF RAW AND DECELLULARISED PORCINE TUNICA VAGINALIS AS A NOVEL ACELLULAR MATRIX(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2021-12-30) ABHIN RAJ K.P.; Sreeranjini A.RThe current research was aimed to develop a novel acellular matrix from porcine tunica vaginalis by a standardised decellularisation protocol and to study the structural and physico-chemical properties of raw tunica vaginalis and the developed acellular matrix. Due to the intimate association of visceral layer of tunica vaginalis with testicular capsule, parietal layer of tunica vaginalis (PTV) were collected for the development of the acellular matrix from the testes of six adult healthy pigs slaughtered at the Meat Technology Unit, College of Veterinary and Animal Sciences, Mannuthy. Grossly PTV was found as an extensive, thin, elastic, flask shaped serous sac with numerous blood vessels and nerves. It lined the scrotum and the inguinal canal and was attached to the tail of epididymis and vas deferens. Histologically PTV comprised mesothelium and dense regular connective tissue composed of thick collagen bundles, a few elastic, reticular and smooth muscle fibres, blood vessels, and nerves. PTV was decellularised by two protocols using aqueous solution of bovine bile and Trypsin EDTA. After decellularisation, physico-chemical properties of the acellular matrix were evaluated and compared with that of raw PTV. Thickness, tensile strength, collagen content and resistance to collagenase enzyme of the decellularised matrix were less when compared with the raw PTV, whereas the collagen solubility was higher. Efficiency of decellularisation was assessed by routine and special histological staining methods, nuclear staining using 4, 6-Diamidino-2-phenylindole dihydro chloride, estimation of glycosaminoglycans, analysis of DNA content and by scanning electron microscopy. Lack of cellularity, lowest DNA content (<50 ng/µL) and increased porosity of acellular matrix revealed that efficiency of decellularisation using Trypsin-EDTA was better than that with bile treatment.ThesisItem Open Access FACTORS OF ANATOMICAL AND GENETIC ORIGIN PREDISPOSING TO INFERTILITY IN CROSSBRED DAIRY COWS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR -, 2021-03-08) ANNIE V. RAJ; K.M. LucyFactors of anatomical and genetic origin predisposing to infertility in crossbred dairy cows were studied using the genitalia collected from 100 dairy cows / heifers from the Meat Technology Unit, Mannuthy. This included six animals culled on account of factors other than infertility with normal reproductive system (control group) and the remaining animals with a known history of infertility. Morphology, morphometry, histology and histochemistry of genitalia, immunohistochemistry of IGFBP-2, scanning electron microscopic peculiarities of various regions of affected genitalia, biochemical analysis of follicular fluid and validation of SNPs of candidate genes were studied. Standard procedures were adopted for histoarchitectural and histochemical studies. Among the group of 100 animals under study, 14 animals showed anatomical defects in the genitalia. Considering the total number of female animals maintained in the farms, the overall incidence of anatomical abnormalities of genitalia was estimated to be 1.24 per cent. Crossbred Holstein Friesian showed a higher incidence of anatomical defects in the genitalia followed by Frieswal and crossbred Jersey. Cervical abnormality was the most common type noticed followed by ovarian hypoplasia, Mullerian duct abnormalities viz., bicornis bicorpus unicollis, uterus unicornis and tubal obstruction. Histologically, fibrous tissue proliferation was the characteristic feature of hypoplastic ovary with the absence of cyclical structures. In the genitalia with tubal obstruction, lamina epithelialis was detached from the mucosa to form clumps in the lumen. The tunica muscularis was approximately twice thicker than the normal oviduct. In uterus unicornis condition, the ovaries showed degenerated primary follicles and degenerating corpora lutea with hyalinised masses. Left uterine horn was absent and in its position, a membranous structure was seen. Right uterine horn revealed an unusual architecture. In the case of bicornis bicorpus unicollis, all the regions were normal. In kinked cervix condition, the cervical wall showed abundance of fibrous tissue and signs of degeneration. Histochemically, carbohydrates, lipids and phosphatases were demonstrated in different regions of the affected genitalia. IGFBP-2 was localized in the primordial and atretic follicles in the hypoplastic ovary. In uterus unicornis condition, invading granulosa cells into the regressing CL and atretic follicles in the cortical region showed a highly positive immunoreaction. Scanning electron microscopic structure of different regions of genitalia with anatomical defects was also explored. Biochemical analysis of follicular fluid revealed that the mean concentrations of potassium, triglycerides, urea, nitrogen, cholesterol, copper and magnesium were higher in group 2 animals. Single nucleotide polymorphisms reported in the study population by ddRADseq in four candidate genes viz., wnt7a, emx2, hoxa13 and lhx1 affecting the development of female reproductive tract were selected for validation. To identify the single nucleotide variations in bovine wnt7a, PCR was performed and the sequence analysis of 273 bp amplicon revealed the targeted SNP at position 89 (A→G transition) and three novel SNPs at positions 119 (A→G transition), 195 (C→ T transition) and 196 (T→A transversion). For identifying the SNP in bovine emx2, PCR - RFLP was performed and confirmed the population as homozygous. To identify the SNP in bovine hoxa13 and lhx1, HRM was performed and did not show any polymorphism. Amplification of exon 2 of foxa2 by PCR revealed polymorphism at 168th position (T→C transition) of the 320 bp product. This highlights the vital role of wnt7a in regulation of reproduction in bovines. The information gathered by this study will provide insights to the extent and causes of infertility among crossbred dairy cattle of Kerala. Biochemical analysis of follicular fluid is a potent tool for assessing reproductive status. The candidate gene wnt7a can be used for genome-wide scans of anatomical abnormality in bovine species at early stages itself so that the route cause can be eliminated by culling such animals. The findings will also be useful for diagnosing and minimizing the anatomical causes of reproductive disorders so that conception rate of the herd can be increased thus reducing economic loss to the farmers.ThesisItem Open Access HISTOMORPHOLOGY AND PHYSICO-CHEMICAL EVALUATION OF RAW AND DECELLULARISED TUNICA VAGINALIS OF BUFFALO FOR USE AS A BIOMATERIAL IN REGENERATIVE MEDICINE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2021-09-30) LHINGNEIVAH DOUNGEL; Indu V RajThe present study was undertaken to develop an acellular matrix from buffalo bull tunica vaginalis by a newly standardised decellularisation technique. Ten pairs of testes were collected hygienically from ten healthy adult Murrah buffalo bulls slaughtered at the Meat Technology Unit, College of Veterinary and Animal Sciences, Mannuthy. For decellularization, the fresh parietal layer of tunica vaginalis removed from the testes samples were processed as per the newly standardised protocol using trypsin-EDTA with triton-X-100 as detergent. The histomorphological, ultrastructural and physico-chemical characteristics of the raw and decellularised tunica vaginalis were compared and efficiency of decellularisation was evaluated by histological staining techniques, total DNA quantification and scanning electron microscopy. It was observed that the average weight of the left testis in adult Murrah buffalo bulls with and without parietal layer of tunica vaginalis (PTV) was more than that of the right testis and exhibited a statistically significant difference. The outer parietal layer of tunica vaginalis that lined the scrotum could easily be separated out from the testis. The vertical span of PTV of left testis was more than that of the right and the difference was statistically significant. Histologically the PTV consisted of a thin mesothelium and dense regular connective tissue made up of thick collagen bundles, a few elastic fibres and reticular fibres supported by blood vessels, smooth muscle fibres and nerves. It was observed that the decellularised PTV were devoid of cells and genetic materials such as DNA while maintaining its overall tissue histoarchitecture. The physico-chemical characteristics like mean thickness, tensile strength and collagen concentration of decellularised buffalo PTV samples were significantly reduced while the collagen solubility per cent and percentage weight loss after collagenase degradation were significantly higher in decellularised PTV than raw PTV samples. It was concluded that the samples of parietal layer of tunica vaginalis of buffalo prepared by using the newly standardised decellularisation technique showed acceptable physico-chemical properties required for a biologic scaffold. It may be used as a biomaterial in regenerative medicine after extensive clinical screening.
