Studies on Fertility Associated Factors and Mx2 Gene Expression for Early Pregnancy Diagnosis

Abstract

Present experiment was carried out in 20 Sahiwal cows to study various fertility associated factors including rheological factors, subclinical endometritis, progesterone concentration, microbiological status of cervico-vaginal mucus and to study the expression pattern of MX2 gene for early pregnancy diagnosis. The study of rheological properties of cervico vaginal mucus included the study of consistency (thin and thick), transparency (Transparent and cloudy), arborization pattern (typical and atypical), pH and spinnbarkeit value. The percent animals with thin and thick consistency were recorded to be 60% and 40%, respectively which were associated with 41% and 25% conception rates. The transparency of cervico vaginal mucus revealed 65 % animals with transparent and 35% with cloudy cervico vaginal mucus discharge with a respective conception rate of 46.15% and 14.28%. In the present study, the percentage of animals revealing typical fern pattern was quite higher (70%) as compared to those displaying atypical fern pattern (30%) and upon observing the conception rate, the cattle having typical arborization pattern (42.85 per cent) had significantly higher (P<0.05) conception rate than the cattle having atypical arborization pattern (16.66 per cent). It was observed that the pH and spinnbarkeit values of cervico-vaginal mucus in conceived cattle (8.36 ± 0.47 and 11.95 ± 1.57 cm) were significantly (P<0.05) higher as compared to those of non-conceived cattle (7.03 ± 0.73 and 8.32 ± 1.13 cm) and were associated with significantly higher conception rates. The screening of experimental animals for subclinical endometritis by white side test revealed that 50% animals tested negative whereas the incidence of mild, moderate and severe form of subclinical endometritis was recorded to be 20%, 15% and 15%, respectively. In endometrial cytology, the percentage of positive animals (55%) was more than that were diagnosed with white side test (50%) indicating higher efficacy of endometrial cytology in diagnosing subclinical infections. The conception rates were finally compared with these results and higher conception rate of 66.67% (06/09) was recorded among the group negative for subclinical endometritis and low conception rate of 9.09% (01/11) was recorded among the infected animals (9.09%). During the study period none of the cows exhibited any overt clinical signs of diseases but the cervico vaginal mucus was taken for microbiological study. The average bacterial load of cervico-vaginal mucus discharge of the conceived animals in preceding oestrus was 2.53X107, which was found to be lower than those of non-conceived animals (2.375 X108). The most frequently isolated bacterium was E. coli (40%) followed by Staphylococcusspp. (25%) and Candidaspp. (10%) after observing their growth on differential media, selective media and their IMViC patterns. The isolates were subjected to genus specific PCR and showed the 556 bp. The antibiotic sensitivity test revealed that E. coli isolates were found to be 100 % sensitive to Amoxycillin sulbactam, Ciprofloxacin, Gentamycin, Levofloxacin, Nitrofurantoin and highly resistant to Tetracycline (62.5 %), Cefixime (37.5%), Nalidixic acid (37.5%), Co-trimoxazole (25%) and the least resistance was observed against Oxytetracycline (12.5%). The Staphylococcus spp. displayed high sensitivity (100%) against Enrofloxacin (EX), Gentamycin (GEN), Levofloxacin (LE) and Ofloxacin (OF) and high resistance to Cephoxitin (100.0%) followed by Doxycycline (62.5%), Streptomycin (37.5%), Oxytetracycline (37.5%) and Amoxicillin Clavulanic acid (37.5%). The antifungals of the Candida spp. the Amphotericin B (AP), Colistin (CL), Itraconazole (IT) and Polymixin (PB) were found to be 100% sensitive followed by Fluconazole (50.0%). The isolates were found highly resistant to Bacitracin, Linezolide, Nalidixic acid, Nitrofurantoin (100 %), followed by Fluconazole (50.0%). The values of plasma progesterone (ng/ml) obtained among conceived vs non-conceived cattle on days 7 (2.26 ± 0.19 vs 1.55 ± 0.09), 17 (5.42 ± 0.35 vs 4.14 ± 0.14) and 21 (6.38 ± 0.39 vs 0.81 ± 0.06) showed a highly significant increase (P<0.01) in amount of progesterone concentration in conceived animals on ascending days while, sudden decrease was observed in the progesterone concentration after day 17 of insemination in non-conceived dairy cattle. Real-Time PCR of MX2 gene may act as biomarker for early pregnancy diagnosis in cattle. The result of MX2 gene mRNA transcript analysis by Real-Time PCR showed a 9.51 to 28.64 fold higher expression in pregnant animals in comparison to non-pregnant animals