Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis).

BACKGROUND/AIMS: Thrombospondins (TSPs) are large multi-modular proteins, identified as natural angiogenesis inhibitors that exert their activity by binding to CD36 and CD47 receptors. The anti-angiogenic effect of TSPs in luteal regression of water buffalo has not been addressed. The present study characterized the expression pattern and localization of TSPs and their receptors in ovarian corpus luteum during different stages of development in buffalo. This study also elucidated the effect of exogenous Thrombospondin1 (TSP1) or the knocking out of the endogenous protein on luteal cell viability and function. Further, the in vitro transcriptional interaction of TSP1 with hormones, LH, PGF2α and angiogenic growth factors, VEGF and FGF2 were also evaluated. METHODS: First, the CLs were classified into four groups based on macroscopic observation and progesterone concentration. mRNA expression of examined factors was measured by qPCR, localization by immunoblotting and immunohistochemistry. TSP1 was knocked out (KO) in cultured luteal cells isolated from late luteal stage CLs (day 1116) by CRISPR/Cas9 mediated gene editing technology in order to functionally validate the TSP1 gene. Isolated cells from late stage CLs were also stimulated with different doses of TSP1, LH, PGF2α, VEGF and FGF2 for various time intervals to determine transcriptional regulation of thrombospondins. RESULTS: mRNA expression of TSPs and their receptors were found to be significantly higher in late and regressed stage of CL as compared to other groups which was consistent with the findings of immunoblotting and immunolocalization experiments. It was observed that TSP1 induced apoptosis, down regulated angiogenic growth factors, VEGF and FGF2 and attenuated progesterone production in cultured luteal cells. However, knocking out of endogenous TSP1 with CRISPR/Cas9 system improved the viability of luteal cells, progesterone synthesis and upregulated the expression of VEGF and FGF2 in the KO luteal cells. PGF2α induced the upregulation of TSPs and Caspase 3 transcripts, whereas treatment with LH and angiogenic growth factors (VEGF and FGF2) down regulated the TSP system in luteal cells. CONCLUSION: Collectively, these data provide evidence that thrombospondins along with their receptors are expressed at varying levels in different stages of CL progression with maximum expression during the late and regressing stages. These results are consistent with the hypothesis that thrombospondins stimulated by PGF2α plays an essential modulatory role in bringing about structural and functional luteolysis in buffalo.

Effect of FecB status on the allometric measurements and reproductive performance of Garole × Malpura ewes under hot semi-arid environment.

The study was conducted with the primary objective to establish the influence of FecB gene mutation on the growth and reproductive performance of Garole × Malpura ewes. The duration of the study was for 5 months involving 97 adult Garole × Malpura (GM) ewes. These 97 animals were segregated based on FecB genotype and the animals were divided into three groups as group I (FecB(BB) ; n = 13), group II (FecB(B+); n = 65), and group III (FecB(++); n = 19). All the animals were kept under semi-intensive management system by subjecting the animals to grazing during day time and housing them in the night. Apart from grazing, ewes were provided with concentrate mixture at the rate of 150 g/day/ewe. The ewes in estrus after 12 h of onset of estrus were hand mated twice at 12-h intervals with a ram of proven fertility in the morning and evening. The pregnancy was confirmed by real time B-mode transabdominal ultrasonography. The experiment was continued until lambing. Under the treatments of different FecB genotype, the ewe's growth and reproductive parameters were assessed. FecB genotype significantly (P < 0.05) influenced body weight, heart girth, pin shoulder length, height at wither, lambing percentage, litter size, twinning percentage, and birth weight of lambs. From this study, it can be concluded that breeding Galore and Malpura breed shall help insure an economically viable production system.

Effect of multiple stresses on growth and adaptive capability of Malpura ewes under semi-arid tropical environment.

A study was conducted to assess the effect of multiple stresses (thermal, nutritional, and walking stress) on the adaptive capability of Malpura ewes in terms of changes in physiological mechanisms and blood biochemical changes. The study was conducted for a period of 35 days covering two estrous cycles during summer season (April-May). The ewes were randomly allocated into two groups of 12 animals each, viz., GI (n = 12; control) and GII (n = 12; multiple stresses). GI ewes were maintained in the shed while GII ewes were subjected to multiple stresses. GI ewes were maintained in shed while GII ewes were subjected to heat stress by exposing them to 32-44 °C (average 42 °C) and RH of 12-26 % for 6 h from 10:00 to 16:00 h in natural environment. GI ewes were provided with ad libitum feeding while GII ewes were provided with restricted feed (30 % of intake of GI ewes) to induce nutritional stress. Further, GII ewes were subjected to walking stress by forcing them to walk 14 km in two spans between 9:00 and 10:30 h and 15:00 and 16:30 h. Blood collection was done at weekly intervals. Multiple stresses significantly (P < 0.01) affected body weight, respiration rate, pulse rate, rectal temperature, sweating rate, tri-iodo-thyronine, thyroxine, cortisol, hemoglobin, packed cell volume, glucose, and total protein. The findings from this experiment provide useful information to understand the ideal nutrient requirement for these animals to adapt to such stresses in the semi-arid tropical environment without compromising production.

Adaptability and growth of Malpura ewes subjected to thermal and nutritional stress.

A study was conducted to assess the effect of combined stresses (thermal and nutritional) on physiological adaptability and growth performance of Malpura ewes. Twenty-eight adult Malpura ewes (average BW 33.56 kg) were used in the present study. The ewes were divided into four groups, viz., GI (n=7; control), GII (n=7; thermal stress), GIII (n=7; nutritional stress), and GIV (n=7; combined stress). The animals were stall-fed with a diet consisting of 60% roughage and 40% concentrate. GI and GII ewes were provided with ad libitum feeding, while GIII and GIV ewes were provided with restricted feed (30% intake of GI ewes) to induce nutritional stress. GII and GIV ewes were kept in climatic chamber at 40 °C and 55% RH for 6 h/day between 1000 and 1600 hours to induce thermal stress. The study was conducted for a period of two estrus cycles. The parameters studied were feed intake, water intake, physiological responses (viz., respiration rate, pulse rate, and rectal temperature), body weight, and body condition scoring (BCS) of ewes. Both thermal and combined stress significantly (P<0.05) affected the feed intake, water intake, respiration rate, and rectal temperature. The feeding schedule followed in the experiment significantly (P<0.05) altered the body weight and BCS between the groups. The results reveal that when compared with thermal stress, nutritional stress had less significant effect on the parameters studied. However, when both these stresses were coupled, it had a significant influence on all the parameters studied in these ewes. It can be concluded from this study that when two stressors occur simultaneously, the total cost may have severe impact on biological function.

Supplementation of vitamin E, selenium and increased energy allowance mitigates the transition stress and improves postpartum reproductive performance in the crossbred cow.

Dairy cow undergoes tremendous physiological challenges during the transition period leading to negative energy balance (NEBAL), impaired immunity and oxidative stress that ultimately compromises the postpartum fertility. Accordingly, we investigated the effects of antioxidant supplementation and increased energy allowance on transition stress and fertility of crossbred cow. Advanced pregnant crossbred cows (n = 26) of 2-4 parity and lactation potential of >10 L/day were divided into two equal groups (n = 13 cows/group). Cows were fed diets either (i) supplemented with vitamin E (80 IU/kg DM), Selenium (Se; 0.3 mg/kg DM) and increased energy allowance in the form of 20% additional concentrate (TRT) or (ii) basal diet without any supplementation as control (CON). Vitamin E and Se were supplemented with wheat flour bolus from -4 to 8 week of calving whereas energy allowance was increased from 2 to 8 week of calving on daily basis to individual animal (where 0 is day of calving). Blood samples were collected on weekly interval from -4 to 8 week of calving. Oxidative stress was assessed by estimation of malondialdehyde (MDA), total antioxidant capacity (TAC), superoxide dismutase (SOD) and catalase (CAT). Immunity level was assessed via estimation of phagocytic activity (PA) of granulocytes and lymphocyte proliferation assay (LPA). Postpartum fertility was assessed by interval to first postpartum estrus (day) and pregnancy rate. Cows in TRT had a significantly lower MDA, higher TAC and decreased activity of SOD and CAT than that of CON (P < 0.05). Phagocytic activity increased at -1, 0 and 3-8 weeks postpartum (P < 0.05) while LPA showed difference (P < 0.05) at parturition, week 4 and 8 postpartum. Marked improvement in the fertility was recorded in terms of early resumption of postpartum estrus (P < 0.001) and higher pregnancy rate (P < 0.05).