Isolation, in vitro expansion and characterization of ovine fetal adnexa-derived mesenchymal stem cells reveals a source dependent trilineage differentiation and growth kinetics.

This study was designed to isolate, cultivate, characterize and evaluate the growth kinetics of mesenchymal stem cells (MSCs) derived from fetal adnexa of sheep. The gravid uteri of ewes were collected from a local abattoir. The MSCs isolated from different fetal regions (Wharton's Jelly [oWJ], cord blood [oCB], amniotic fluid [oAF] and amniotic Sac [oAS]) were expanded in vitro and characterized for surface and pluripotency markers. The growth kinetics of MSCs was compared at 3rd and 5th passages. Similarly, the colony-forming efficiency (CFE) assay was performed at 3rd passage. The fetal adnexa-derived ovine MSCs showed the expression of CD73, CD90 and CD105. Similarly, the MSCs also expressed pluripotency markers, OCT4 and SOX2. Besides, cells also differentiated into osteogenic, chondrogenic and adipogenic lineages. The MSCs in culture showed a typical growth curve with initial lag phase, an exponential phase, a plateau phase and a decline phase. The growth rate was highest in oAF-MSCs at P5. The population doubling time (PDT) was highest in oAS-MSCs (87.28 ± 3.24 h), whereas the colony number was highest in oAF-MSCs (53.67 ± 4.06). The study reveals that oAF-MSCs were superior which outperformed other MSCs indicating that oAF-derived MSCs could be utilized for regenerative medicine.

Interferon-tau stimulated gene expression: A proxy to predict embryonic mortality in dairy cows.

The embryonic mortality in cows is a growing concern for an ever-expanding dairy industry. The current study was an attempt to shorten the open period of dairy cows having suffered embryonic loss by diagnosing them at an earlier stage. The blood samples were collected from the Karan Fries (KF) cows on days 0 (day of AI/estrus), 4, 8, 12, 14, 16, 18, 21, 24, 28, 35 and 42 post insemination. The experimental animals were then categorized into pregnant (P), conception failure/early embryonic mortality (EEM) and late embryonic mortality cows (LEM), based on progesterone assay, ultrasonography and per-rectal palpation. There were 6 animals in each group. The plasma progesterone was higher in pregnant than EEM and LEM cows. Plasma Interferon-tau concentration was significantly (p < 0.05) lower in LEM than pregnant cows where it could be detected from day 14-21 but was non-detectable in EEM cows. The mRNA expression of ISG15, OAS1, MX1 and MX2 in blood neutrophils was significantly (p < 0.05) higher from day 8-42 as against day 0 in pregnant cows. The highest expression was observed around day 18-21 in pregnant cows. The ISG15, OAS1, MX1 and MX2 mRNA expression was significantly (p < 0.05) higher from day 4-42 as compared to day 0 in LEM cows, whereas in EEM cows the expression stayed close to that of day 0 (1.00 ±â€¯0.00). The mRNA expression of ISG15, OAS1, MX1 and MX2 started to decline from day 24 onwards. The degree of expression of Interferon-tau stimulated genes was higher in pregnant and LEM cows than EEM cows. The study reveals that the Interferon tau stimulated gene expression in neutrophils can act as peripheral biomarkers for detecting the embryonic mortality in dairy cows.

Effect of tropical thermal stress on peri-implantation immune responses in cows.

Pregnancy losses during the peri-implantation period cause a negative impact on the reproductive and economic performance of dairy herds. In this study, we investigated the possible immunological factors which may contribute to pregnancy loss during the peri-implantation period under different seasons of tropical conditions. Eighteen Karan Fries cows, six cows in each season (W: winter; HH: hot-humid; HD: hot-dry) were selected. These cows exhibited heat and were brought for artificial insemination (AI; day 0). Blood was collected on days 0, 10, 14, 16, 18, 21 and 28 post-AI. Pregnancy was confirmed by non-return to heat, progesterone assay and ultrasonography. Blood neutrophils were isolated and tested for their number, myeloperoxidase (MPO) concentrations and expression of cell adhesion molecules (CD11b, CD14, CD25, CD47), interferon tau stimulated genes (ISG15, MX1, OAS1) and chemokine receptors (CXCR1, CCL2). Plasma cortisol, progesterone, IL-2 and IL-10 were also estimated. Neutrophil number, MPO levels, the relative expression of various neutrophil receptors and plasma IL-2 were low between days 14-21 post-AI in all seasons. However, plasma cortisol and IL-10 were higher during the same period. The inflammatory activity of neutrophils, plasma IL-2 and cortisol were highest in HH, intermediate in HD and lowest in W season. However, plasma progesterone and IL-10 were highest in W season and lowest in HH season. Our results show that blood neutrophils sense the implanting embryo and downregulate their activity to ensure successful implantation; however, under harsh environmental conditions, it is a great challenge for the immune system to maintain such balance and thus it may negatively affect the outcome of pregnancy.

JAK3 and PI3K mediate bovine Interferon-tau stimulated gene expression in the blood neutrophils.

Interferon tau, a 23 kDa trophoblast derived protein diffuses out from the uterus into the circulation and leads to the expression of IFNτ stimulated genes viz. ISG15 and OAS1 in blood neutrophils. The IFNτ pathway is species as well as tissue specific. To unsnarl the IFNτ downstream signaling pathway, the blood neutrophils were incubated simultaneously with 10 ng/ml of recombinant bovine interferon tau and the inhibitors of JAK2 (AG490), JAK3 (CP690550), p38 (SB202190), PI3K/Akt (LY294002), and MAPK/Erk (U0126) at specific doses for 4-hr duration. The IFNτ pathway was determined through real-time gene expression of ISG15 and OAS1; immunocytochemistry of ISG15; and Western blotting of ISG15, OAS1, pJAK3 and PI3K. The ISG15 and OAS1 expression decreased significantly (p < 0.001) in the presence of pJAK3 and PI3K inhibitors as compared to a positive control where only interferon tau was used. Immunocytochemistry revealed an attenuated ISG15 response while stimulating blood neutrophils with pJAK3 inhibitor (CP690550) and PI3K inhibitor (LY294002). Similarly, Western blot analysis of neutrophil protein fraction showed weak signals of ISG15, OAS1, pJAK3 and PI3K in the presence of pJAK3 and PI3K inhibitors. The expression profile, immunocytochemistry and western blot analysis revealed a JAK3 and PI3K mediated interferon-tau stimulated gene expression in blood neutrophils.

PCR-RFLP based genotyping of Indian isolates of Toxoplasma gondii.

Toxoplasma gondii is an apicomplexan parasite capable of infecting a wide variety of warm-blooded animals, including birds and humans and is zoonotically important too. Felidae serve its definitive hosts and most infections are inoccous while in various intermediate hosts (e.g. sheep), it is responsible for abortion, still births. Humans which are immune compromised are also susceptible to toxoplasmosis. Most of the epidemiological studies have revealed it to be belonging to three clonal types with exceptions in South Africa having atypical isolates. Current genotyping was carried out at 11 genetic loci (SAG1, 5'-SAG2, 3'-SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358 and PK1) using multiplex-nested polymerase chain reaction restriction fragment length polymorphism (Mn-PCR-RFLP). SAG1, alt SAG2, SAG3, BTUB, GRA6, C22-8, C29-2, L358 and PK1 could differentiate our strain/isolates as type I (T. gondii RH) and type III (T. gondii isolates from Chennai and Izatnagar). 5'SAG2 and 3'SAG2 in combination confirmed these as above mentioned genotypes. Further, the T. gondii RH was assigned Toxo DB#10 and local isolates of T. gondii were assigned Toxo DB#2. The present study is the first report on existence of Type III T. gondii lineage from animal population of Indian subcontinent based on PCR-RFLP.

Inorganic zinc supplementation modulates heat shock and immune response in heat stressed peripheral blood mononuclear cells of periparturient dairy cows.

Thermal stress in India is one of the major constraints affecting dairy cattle productivity. Every attempt should be made to ameliorate the heat and calving related stress in high producing dairy cows for higher economic returns. In the current study, inorganic zinc was tried to alleviate the adverse effects of thermal stress in periparturient cows. Twelve cows, six each of Sahiwal and Karan Fries (KF) in their second parity with confirmed pregnancy were chosen for the experiment. The blood samples were collected periparturiently on three occasions viz. -21, 0 and +21 days relative to calving. The in vitro study was conducted after isolating peripheral blood mononuclear cells (PBMC) from whole blood. The cultured PBMC were subjected to three different levels of exposures viz. 37°C as control, 42°C to induce thermal stress and 42°C + zinc to ameliorate the adverse effects of high temperature. Heat shock lead to a significant (P<0.05) rise in the level of heat shock proteins (HSP). HSP was more on the day of calving as well. KF showed more HSP concentration than Sahiwal breed indicating the heat bearing capacity of later. Zinc treatment to thermally stressed PBMC caused a fall in the HSP concentration in both the breeds during periparturient period. Moreover, heat stress increased significantly (P<0.05) the Interleukin 6 (IL-6) concentration which declined upon zinc supplementation to PBMC. IL-6 levels decreased periparturiently. Heat and calving related stress caused a fall in the IL-12 levels which increased significantly (P<0.05) with zinc supplementation. These findings suggest that zinc supplementation attenuates the HSP response and augments immunity in PBMC of periparturient dairy cows. The study could help to alleviate the heat stress and potentiate immunity by providing mineral supplements in periparturient dairy cattle habituating tropics.

Incidence of mastitis and activity of milk neutrophils in Tharparkar cows reared under semi-arid conditions.

Rearing of indigenous Tharparkar (TP) cows (native of arid Thar deserts) under high humid conditions (>75 % humidity) has increased the incidence of mammary infections in them. A study was undertaken to see the number, activity, and expression of milk neutrophils isolated from healthy and mastitic cows. There was a significant (P < 0.05) influx in milk somatic cell counts (SCC) and neutrophils in sub-clinical and clinical mastitis cows. No change was observed in the phagocytic activity (PA) of milk neutrophils between healthy and sub-clinical mastitis (SCM) cows, but these activities decreased significantly (P < 0.05) in clinical cases. Chemotactic activity showed a significant difference between all the groups. Lactose varied significantly (P < 0.05) between healthy, sub-clinical, and clinical mastitis (CM) cows. Expression of chemokine receptor (CXCR1) was more in mastitis cows and also higher as compared to CXCR2. No change was observed in cluster of differentiation molecule (CD62L) among all the three groups of TP cows. Expression of interleukin (IL-8) and CD11b was low in healthy cows, increased significantly (P < 0.05) in both sub-clinical and mastitis cows. This study indicates that low producing TP cows are also prone to mammary infections when reared under semi-arid conditions.

Effect of in vitro zinc supplementation on HSPs expression and Interleukin 10 production in heat treated peripheral blood mononuclear cells of transition Sahiwal and Karan Fries cows.

The changing climatic scenario with apprehended rise in global temperature is likely to affect the livestock adversely vis-à-vis production and reproduction. This has prompted more focus in addressing the unfavorable effects of thermal stress in livestock system. Presuming that the trace element zinc is indispensible for cellular antioxidant system and immune function, the present study was designed to investigate the effect of zinc treatment on heat stress alleviation and immune modulation in peripheral blood mononuclear cells (PBMC) of indigenous and crossbred transition cows. Twelve cows, six each of Sahiwal and Karan Fries (KF) in their second parity with confirmed pregnancy were selected for the experiment. The blood samples were collected at -21, 0 and +21 days in relation to expected date of calving. The experiment was carried out in vitro after isolating PBMC from whole blood. The 48h cultured PBMC were subjected to assorted levels of exposures viz. 37°C, 42°C to impose heat stress and 42°C+zinc to alleviate heat stress and modulate immunity. The PBMC viability was 86%, 69% and 78%, respectively. The mRNA expression of heat shock proteins (HSP 40, 70 and 90α) and Interleukin-10 (IL-10) production varied between the two breeds vis-à-vis days and levels of exposure. The mRNA expression of HSP40 and HSP70 was significantly (P<0.05) higher in Karan Fries than the Sahiwal cows. Both the breeds showed maximum expression of HSP on the day of parturition, more so in KF than Sahiwal. There was a significant (P<0.05) difference in the HSP mRNA expression at different levels of exposure. Zinc treatment to heat stressed PBMC caused a significant (P<0.05) down regulation of HSP. For immune status, anti-inflammatory cytokine, IL-10 in the culture supernatant was accessed. The IL-10 was significantly (P<0.05) higher in Karan Fries (168.18±14.09pg/ml) than the Sahiwal cows (147.24±11.82pg/ml). The IL-10 concentration was highest on the day of calving. Zinc treatment reduced the IL-10 concentration. From the study, it could be concluded that the zinc supplementation in heat stressed PBMC can ameliorate thermal stress and modulate immune response which can act as a model for reducing heat stress during the periparturient period in tropical livestock.