FACTORS OF ANATOMICAL AND GENETIC ORIGIN PREDISPOSING TO INFERTILITY IN CROSSBRED DAIRY COWS
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Date
2021-03-08
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR -
Abstract
Factors 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.
Description
Submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy in Veterinary Anatomy and Histology