Thyroid hormone dynamics of Tharparkar and Sahiwal cattle during induced heat stress.

Thyroid hormones and Cortisol level are the essential biomarkers in the assessment of stress condition. This study was done to estimate the metabolic hormonal profile of Tharparkar and Sahiwal during heat stress condition. The experiment was conducted on two groups consisting of Tharparkar and Sahiwal animals (5 in each group) and the experimental period comprised a 7-day acclimatization period, a heat exposure period of 21 days at control (25 °C), moderate (35 °C) and severe (42 °C) heat stress within a 9-10-day recovery period between each exposure. The hormonal concentrations of T3, T4 and cortisol were determined in serum. The serum concentration of Thyroxine (T4) and tri-iodothyronine (T3) decreases whereas cortisol level increases in both the breeds when subjected to heat stress. However, the serum level of T4 was significantly (p < 0.05) more declined in Sahiwal as compared to Tharparkar but there was no significant difference found between the two breeds in serum T3 levels. The cortisol levels were elevated in both breeds during heat stress but significantly (p < 0.05) more elevated in the Sahiwal. Hence, observations of these hormonal profiles suggest a better thermo-adaptability in Tharparkar as compared to Sahiwal.

Evaluation of thermo-adaptability between Tharparkar (Bos indicus) and crossbred (Bos indicus X Bos taurus) calves in a controlled environment.

The global warming driven climate change has increased the susceptibility of livestock around the globe to heat stress (HS), which reduces animal productivity and threatens the sustainability of marginal farmers. The objective of this investigation was to evaluate thermo-adaptability between Tharparkar calves (TC), an indigenous milch breed of India and crossbred calves (CC) during induced heat stress in controlled environment. For this purpose, 12 apparently healthy male calves (six in each group) aged 5-6 months, were selected. The experiment was conducted at physiologically comfortable temperature (25 °C), moderate HS (31 °C) and severe HS (37 °C) for 21 days each in a psychrometric chamber. In each experimental day, the calves were exposed to 6 h of heat. There were 7 days of acclimatization period before experiment and 10 days of recovery period at ambient temperature between each 21 day exposure period. During experimental period, the blood was collected at 1st, 6th, 11th, 16th, 21st day and among ten-day recovery period the blood was collected at 5th day. Physiological responses, serum electrolytes, metabolic enzymes profiles, antioxidant capacity, oxidative stress status and general endocrine milieu were studied. Relative mRNA expression study of Heat Shock Protein (HSP) 70, HSP90, induced Nitric Oxide Synthase (iNOS) and endothelial NOS (eNOS) were carried out by qPCR. There was significant (p < 0.05) change in the displacement in rectal temperature, respiration rate, serum alanine aminotransferase level between two breeds at moderate and severe HS. Similar change was observed in total antioxidant capacity, superoxide dismutase, and endocrinological parameters. The comparatively lower mRNA expression of HSP70 and higher expression of HSP90 in TC than CC point the better thermo-adaptability of the same. The results of the experiment indicated that TC are more thermo-adaptable than CC at different modality of stress in controlled temperature conditions.

Effect of milking environment enrichment through music on production performance and behaviour in cattle.

Enrichment of milking environment through music has been proposed to help animals to cope with divergent stressors. In sight of the above, a study was conducted to evaluate the effect of Indian instrumental music-based environmental enrichment played in yaman raga on milk production performance and behaviour in cattle. A total of 21 lactating dairy cattle (Vrindavani crossbred cows) having similar parity and stage of lactation were selected in three groups - T1, T2 and T3, each consisting of seven animals. The T1 and T2 groups were exposed to instrumental flute and sitar, respectively, 10 min prior to the start of milking and continued till completion of milking; while the T3 group served as control. Musical enrichment of the environment was done using recorded-tape of flute and sitar was played in yamen raga at 40-60 (dB) decibel intensity. The results revealed a non-significant difference in milk yield, rectal temperature, respiration rate, T3 (triiodothyronine) and T4 (thyroxine) hormones. However, there exhibited a significant (p < 0.05) difference in milking time, milking speed, cortisol hormones and behavioural parameters such as milk let-down in the animals exposed to music compared to the control group. Thus, the results have significant implications relating to the behavioural fitness and welfare of dairy animals and reducing residual milk.

Alpha lipoic acid supplementation ameliorates the wrath of simulated tropical heat and humidity stress in male Murrah buffaloes.

A supplement which ameliorates temperature-humidity menace in food producing livestock is a prerequisite to develop climate smart agricultural packages. A study was conducted to investigate the heat stress ameliorative efficacy of alpha lipoic acid (ALA) in male Murrah water buffaloes (Bubalus bubalis). Eighteen animals (293.61 ± 4.66Kg Bwt) were randomly allocated into three groups (n = 6); NHSC (non-heat-stressed control), HS (heat-stressed) and HSLA (heat-stressed-supplemented with ALA@32 mg/kg Bwt orally) based on the temperature humidity index (THI) and ALA supplementation. HS and HSLA were exposed to simulated heat challenge in a climatically controlled chamber (40 °C) for 21 consecutive days, 6 h daily. Physiological responses viz. Respiration rate (RR), Pulse rate (PR) and Rectal temperature (RT) were recorded daily before and after heat exposure. Blood samples were collected at the end of heat exposure on days 1, 6, 11, 16, and 21 and on day 28 (7th day post exposure which is considered as recovery) for peripheral blood mononuclear cells (PBMCs) separation, followed by RNA and Protein extraction for Real time quantitative PCR and Western blot analysis respectively, of heat shock proteins (HSPs). Two-way repeated measure ANOVA was performed between groups at different experimental periods. RR (post exposure) in HS and HSLA was significantly higher (P < 0.05) than NHSC from day 1 onwards but HSLA varied significantly from the HS 8th day onwards. Post exposure RT and PR in both HS and HSLA varied (P < 0.05) from NHSC throughout the study; but between HS and HSLA, RT significantly varied on initial 2 days and last 6 days (from days 16 to 21). HSP70 mRNA expression significantly up regulated in high THI groups with respect to the low THI group throughout the experimental period. During chronic stress (days 16 and 21) HSP70 significantly (P < 0.05) increased in HS but not in HSLA (P > 0.05) with respect to NHSC. ALA supplementation up-regulates and sustains (P < 0.05) the expression of HSP90 in HSLA in comparison to the HS and NHSC. HSP105 expression was significantly up-regulated (P < 0.05) in HS on days 16 and 21 (during long-term exposure) but only on day 21 (P < 0.05) in HSLA. HSP70, HSP90, and HSP105 protein expression dynamics were akin to the mRNA transcript data between the study groups. In conclusion, supplementing ALA ameliorates the deleterious effect of heat stress as reflected by improved physiological and cellular responses. ALA supplementation improved cellular antioxidant status and sustained otherwise easily decaying heat shock responses which concertedly hasten the baton change from a limited window of thermo tolerance to long run acclimatization.

Regulation of steroidogenic function of luteal cells by thrombospondin and insulin in water buffalo (Bubalus bubalis).

The present study examined the effect of exogenous thrombospondin 1 (TSP1) on the steroidogenic function of luteal cells cultured invitro. Furthermore, the transcriptional interaction of insulin with TSP1 and its receptor, cluster of differentiation 36 (CD36) were also investigated. At the highest dose (500ngmL-1) TSP1 significantly downregulated the expression of the angiogenic marker von Willebrand factor (vWF) and progesterone production in cultured luteal cells. Moreover, the simultaneous upregulation in the expression of caspase 3 by exogenous TSP1 was consistent with a reduction in the number of viable luteal cells as determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltertrazolium bromide (MTT) assay after 72h of culture. However, the expression of critical enzymes in the progesterone synthetic pathway was not significantly modulated by treatment with TSP1 in cultured luteal cells. Knocking out of endogenous TSP1 with the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPRassociated protein9 (Cas9) system improved the viability of luteal cells as well as increasing progesterone production and decreasing caspase 3 activation. Insulin treatment suppressed the expression of TSP1 and CD36 in cultured luteal cells in a dose- and time-dependent manner. To conclude, TSP1 acts as a negative endogenous regulator of angiogenesis that attenuates progesterone production, possibly by reducing the number of luteal cells via apoptosis during luteal regression, whereas insulin as a luteinising signal may have inhibited the thrombospondin system for the efficient development of luteal function.

Transcriptional and translational abundance of Bone morphogenetic protein (BMP) 2, 4, 6, 7 and their receptors BMPR1A, 1B and BMPR2 in buffalo ovarian follicle and the role of BMP4 and BMP7 on estrogen production and survival of cultured granulosa cells.

BMPs and their receptors modulate the granulosa cell (GC) function in the follicle of domestic animals. Since little is known on BMPs in the buffalo, the present study was aimed to investigate the expression of BMP2, 4, 6, 7 and their receptors BMPR1A, BMPR1B, BMPR2 in the GC and theca cells (TC) of ovarian follicles and the role of BMP4 and BMP7 on buffalo GC. Follicles were classified into four groups based on size and E2 level in the follicular fluid as follows: (i) Group1(4-6 mm; <0.5 ng/mL) (ii) Group 2 (7-9 mm; 0.5-5 ng/mL) (iii) Group 3 (10-13 mm; 5-40 ng/mL) and (iv) Group 4 (dominant follicle) (>13 mm; >180 ng/mL). The results revealed that except BMP6, BMP2, 4 7 and receptors BMPR1A, BMPR1B and BMPR2 showed a minimum of 1.5-2 fold increase in mRNA expression in the GC of dominant follicle as compared to other follicle classes. In the dominant follicle, a two-fold increase in BMP4 and BMP7 expression was observed in the TC. At 100 ng/mL, the BMP4 and BMP7 either alone or in combination maximally down-regulated CASPASE3 and stimulated the transcripts of PCNA, FSHR and CYP19A1 that was supported by E2 secretion in the granulosa cell culture suggesting their role in cell survival and E2 production. In conclusion, GC and TC of dominant follicles express BMP 2, 4, 6, 7 and their receptors BMPR1A, BMPR1B and BMPR2. BMP4 and BMP7 stimulate E2 production and promote GC survival.

Expression and molecular cloning of interferon stimulated genes in buffalo (Bubalus bubalis).

Buffalo, the most important livestock species in tropical India, remains to be a poor breeder mainly due to embryonic mortality (65%) occurring mostly between 16 and 18 days of pregnancy. Early and accurate diagnosis of pregnancy can thus become a boon for successful herd management in buffalo. However, most of the currently available methods allow diagnosis only after 30 days post AI. Interferon tau (IFNT), the first pregnancy recognition signal in ruminants is one such molecule, which stimulates expression of various Interferon stimulated genes (ISGs) in the peripheral blood mononuclear cells (PBMC's) concomitant with IFNT signaling which occurs around maternal recognition of pregnancy (MRP). Hence, the study was planned to demonstrate the expression dynamics of ISGs (OAS1, MX1, MX2 and ISG15) in PBMCs during peri-implantation period in buffalo and also molecular cloning and expression of suitable ISG coded protein (s) in suitable host. Blood was collected from two groups of multiparous buffaloes: Group1: (n = 10) inseminated/pregnant (Experimental) and Group2: (n = 10) anestrous/non pregnant (Control). The expression profile of ISGs was then analyzed using real time qPCR. Expression profile of most ISGs was observed to increase through day 14 to day 20 post AI and declined thereafter. On the basis of differential gene expression at day 18 post AI, OAS1 and MX2 were identified as suitable ISG candidate biomarkers for accurate pregnancy diagnosis within 18 days post AI. Molecular cloning and expression of selected ISGs in a suitable prokaryotic expression vector was done thereafter. Bulk expression of the recombinant proteins was done and purified by affinity chromatography and confirmed by Western blot using Mouse Monoclonal His-probe antibodies. To conclude, as OAS1 and MX2, showed distinct differential expression at day 18 post AI, they may serve as ideal biomarkers for detection of early pregnancy in buffalo.

Expression dynamics of HSP90 and nitric oxide synthase (NOS) isoforms during heat stress acclimation in Tharparkar cattle.

Six male Tharparkar cattle of 2-3 years old were selected for the study. After 15-day acclimation at thermoneutral zone (TNZ) in psychrometric chamber, animals were exposed at 42 °C for 6 h up to 23 days followed by 12 days of recovery period. Blood samples were collected during control period at TNZ (days 1, 5, and 12), after heat stress exposure (day 1, immediate heat stress acclimation (IHSA); days 2 to 10, short-term heat stress acclimation (STHSA); days 15 to 23, long-term heat stress acclimation (LTHSA); days 7 and 12, recovery period), and peripheral blood mononuclear cells (PBMCs) were isolated for RNA and protein extraction. The messenger RNA (mRNA) and protein expression in PBMCs were determined by qPCR and western blot, respectively. Samples at TNZ were taken as control. The mRNA expression of HSP90, iNOS, and eNOS was significantly upregulated (P < 0.05) on day 1 (ISHA) as compared to control, remained consistent during STHSA, again increased during LTHSA, and finally reduced to basal level during recovery period. The protein expression of HSP90, iNOS, and eNOS were akin to their transcript pattern. PBMC culture study was conducted to study transcriptional abundance of HSP90, iNOS, and eNOS at different temperature-time combinations. The present findings indicate that HSP90, iNOS, and eNOS could possibly play an important role in mitigating thermal insults and confer thermotolerance during long-term heat stress exposure in Tharparkar cattle.

Expression analysis of Toll like receptors and interleukins in Tharparkar cattle during acclimation to heat stress exposure.

Six male Tharparkar cattle of 2-3 years old were selected for the study. After 15 days acclimation at thermo neutral zone (TNZ) in psychrometric chamber, animals were exposed at 42°C for 6h up to 23 days followed by 12 days of recovery period. Blood samples were collected during control period at TNZ (day 1, 5 and 12), after heat stress exposure (day 1-10, Short Term Heat Stress Acclimation - STHSA; day 15-23, Long Term Heat Stress Acclimation - LTHSA) and recovery period (day 7 and 12) and peripheral blood mononuclear cells (PBMCs) were isolated for RNA and protein extraction. Serum cortisol concentration was assessed by RIA. The mRNA and protein expression in PBMCs were determined by qPCR and western blot respectively. Samples at TNZ were taken as control. Serum cortisol concentration was increased (P<0.05) during STHSA and gradually declined during LTHSA. Toll like receptor 2 (TLR 2) expression was up regulated (P<0.05) during STHSA and declined to basal level during LTHSA and recovery phase. However, toll like receptor 4 (TLR 4) expression was up regulated (P<0.05) during STHSA and LTHSA while declined in recovery phase. Interleukin 2 (IL2) and interleukin 6 (IL 6) were up regulated (P<0.05) during STHSA and reduced to basal level during LTHSA. PBMCs culture study was conducted to study transcriptional abundance of TLR2/4 and IL2/6 at different temperature-time combinations. The present findings indicate that TLR 2/4 and IL 2/6 could possibly play a vital role in thermo tolerance in Tharparkar cattle during short term and long term heat stress exposure.

Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) synergistically promote steroidogenesis and survival of cultured buffalo granulosa cells.

The present study investigated the combined effect of fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGF-A) on estradiol (E2) secretion and relative abundance of mRNA for aromatase enzyme (CYP19A1), proliferating cell nuclear antigen (PCNA) and BCL-2 associated X protein (BAX) in cultured buffalo granulosa cells (GCs). Follicles were isolated and classified into four groups based on size and E2 concentration in follicular fluid (FF): Small, 4-6mm diameter, E2<0.5ng/ml; Medium, 7-9mm, E2=0.5-5ng/ml; Large, 10-13mm, E2=5-40ng/ml; Preovulatory (PFs), >14mm, E2>180ng/ml. The GCs of PF were cultured in 24 well cell culture plates and allowed to become 75-80% confluent. Then cultured GCs were treated with FGF2 (200ng/ml) and VEGF-A (100ng/ml) separately and in combination for three incubation periods (24, 48 and 72h). Estradiol secretion was greater in GCs treated with FGF2+VEGF-A compared to FGF2 or VEGF-A at all incubation periods and was greatest (P<0.05) at 72h of incubation. The relative abundance of CYP19A1 and PCNA mRNA were relatively consistent with the amount E2 secretion. In contrast, the relative abundance of Bax mRNA was less in GCs treated with the combination of FGF2 and VEGF-A as compared to either FGF2 or VEGF-A alone and the least concentration (P<0.05) was at 72h of incubation. Findings with use of immunocytochemistry of cells treated with these factors were consistent to the relative abundance of mRNA transcript for the factor. The present findings indicate that FGF2 and VEGF-A may function in a synergistic manner to promote steroidogenesis and survival of cultured buffalo GCs.

Expression dynamics of HSP70 during chronic heat stress in Tharparkar cattle.

Six male Tharparkar cattle aged 2-3 years were selected for the study. The animals were acclimatized in the psychrometric chamber at thermoneutral zone (TNZ) for 15 days and then exposed to 42 °C temperature up to 23 days followed by 12 days of recovery period. Physiological responses were estimated, and peripheral blood mononuclear cells (PBMCs) were isolated at TNZ on day 1, day 5, and day 12; after 6 h of heat stress exposure on day 16 to day 20, day 25, day 30, day 32, day 34, day 36, and day 38; and a recovery period on day 45 and day 50. The PBMCs were cultured to study the effect of thermal challenge on HSP70 messenger RNA (mRNA) expression pattern at different temperature-time combinations. The mRNA and protein expression of HSP70 in PBMCs along with serum extracellular HSP70 (eHSP70) was increased (P < 0.05) and showed two peaks on day 17 and day 32 (2nd and 17th days of thermal challenge, respectively). The HSP70 mRNA expression was increased (P < 0.05) in a temperature- and time-dependent manner in heat stress challenge treatment as compared to control in cultured PBMCs. HSP70 expression was found to be higher (P < 0.05) after 10 days of heat exposure (corresponds to chronic heat stress) as compared to the first 5 days of heat stress (corresponds to short-term heat stress) and control period at TNZ. The present findings indicate that HSP70 is possibly involved in heat stress adaptive response in Tharparkar cattle and the biphasic expression pattern may be providing a second window of protection during chronic heat stress.

Expression and functional role of Bone Morphogenetic Proteins (BMPs) in cyclical corpus luteum in buffalo (Bubalus bubalis).

The role of growth factors in the modulation of ovarian function is an interesting area of research in reproductive biology. Recently, we have shown the expression and role of IGF, EGF, VEGF and FGF in the follicle and CL. Here, we report the presence of Bone Morphogenetic Proteins (BMPs) and their functional receptors in the corpus luteum (CL) of buffalo. The bubaline CL was classified into four stages according to the morphology and progesterone (P4) concentration. The qPCR, immunoblot and immunohistochemistry studies revealed that BMP2 and BMP Receptors (BMPR1A, BMPR1B and BMPR2) were significantly upregulated during the mid stage whereas BMP4 and BMP7 were upregulated during the early stage of CL (P<0.05). Studies on primary luteal cell culture (LCC) using mid CL showed a significant time and concentration dependent effect of BMP4 and BMP7 (P<0.05). At 100ngml-1, the BMPs maximally stimulated the transcripts of StAR, CYP11A1 and 3ßHSD that paralleled with P4 accretion in the media (P<0.05). Further, the BMP4 as well as BMP7 upregulated the transcripts of PCNA and downregulated CASPASE3 in the LCC at the same concentration (P<0.05). Though the combined effect of BMP4 and 7 was significantly higher (P<0.05) than that of individual one, it was not additive. In conclusion, the expression of BMPs and their receptors were dependent on the stages of CL in the buffalo. Treatment of LCC with BMPs in vitro confirmed the presence of functional receptors that stimulated the P4 production and luteal cell survival. Moreover, the results support the concept that the upregulation of P4 and its biosynthetic pathway enzymes such as CYP11A1, StAR and 3ßHSD in the CL is likely due to the autocrine and /or paracrine effects of BMP4 and BMP7 under physiological milieu.

Modulatory role of leptin on ovarian functions in water buffalo (Bubalus bubalis).

The aim of the present study was to demonstrate the modulatory role of leptin on bubaline granulosa cells (GCs) and luteal cells (LCs) functions using an in vitro cell culture system and to establish a cross talk between leptin and insulin-like growth factor-1 (IGF-1). GCs were collected from group IV follicles (>13 mm size) and LCs from mid-luteal phase corpus luteum and were grown in serum-containing media supplemented with leptin at three different dose rates (0.1, 1, and 10 ng/mL) and time durations (24, 48, and 72 hours). We evaluated the production and secretion of estradiol (E2) and progesterone (P4) using RIA and the mRNA expression of steroidogenic acute regulatory protein (STARD1), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450 aromatase (CYP19A1), sterol regulatory element-binding protein 1 (SREBP1), steroidogenic factor-1 (SF1), anti-apoptotic gene PCNA, pro-apoptotic gene caspase 3 and endothelial cell marker, Von Willebrand factor (vWF), using quantitative real-time polymerase chain reaction. The results depicted a direct inhibitory action of leptin on GCs steroidogenesis in a time-dependent manner (P < 0.05), whereas in the presence of IGF-1 the inhibitory effect was reverted. Furthermore, leptin augmented both cellular proliferation (PCNA) and apoptosis (caspase 3). On the other hand, in LCs, leptin alone showed an apparent stimulatory effect on steroidogenesis (P < 0.05); however, in the presence of IGF-1, an antagonistic effect was witnessed. Moreover, leptin had an inhibitory effect on apoptosis while promoted cellular proliferation and angiogenesis. These findings were further strengthened by immunocytochemistry. To conclude, these observations for the first time reported that in buffaloes leptin has a direct dose-, time-, and tissue-dependent effect on ovarian steroidogenesis, angiogenesis, and cytoprotection, and furthermore, it can regulate the effect of systemic factors like IGF-1. Hence, this in vitro study provides an insight into the putative roles of leptin alone and its interactions in vivo.

A prospective comparative clinical study of peripheral blood counts and indices in patients with primary brain tumors.

BACKGROUND: Elevation of the neutrophil to lymphocyte ratio (NLR) has been shown to be an indicator of poor prognosis in many malignancies including recurrent glioblastoma multiforme. OBJECTIVES: This study was aimed at assessing if the NLR and other leukocyte counts and indices were deranged in treatment-naïve patients with primary brain tumors when compared with an age-matched healthy control group. MATERIALS AND METHODS: This was a prospective comparative clinical observational study by design. A healthy control population was compared with treatment-naïve patients diagnosed with intra- and extraaxial brain tumors. Leukocyte counts (neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts) as well as leukocyte ratios such as the NLR and the monocyte to lymphocyte ratio (MLR) were calculated. We also evaluated if the counts and indices were related to the tumor volume. RESULTS: In all patients with tumors, the platelet and neutrophil counts were elevated when compared to the controls. In contrast, monocyte counts and the MLR were found to be decreased in patients with tumors when compared to the controls. The subset of patients with glioblastoma showed a significant increase in NLR when compared to the controls. CONCLUSIONS: Significant changes in the neutrophil, monocyte, and platelet counts as well as NLR and MLR were observed. Prospective longitudinal studies are required to determine the prognostic and therapeutic implications of these findings.

Impact of heat stress, nutritional restriction and combined stresses (heat and nutritional) on growth and reproductive performance of Malpura rams under semi-arid tropical environment.

A study was conducted to assess the combined effect of heat stress and nutritional restriction on growth and reproductive performances in Malpura rams. Twenty-eight adult Malpura rams (average body weight (BW) 66.0 kg) were used in this study. The rams were divided into four groups: CON (n = 7; control), HES (n = 7; heat stress), NUS (n = 7; nutritional stress) and COS (n = 7; combined stress). The study was conducted for a period of 2 months. CON and HES rams had ad libitum access to their feed while NUS and COS rams were under restricted feed (30% intake of CON rams) to induce nutritional stress. The HES and COS rams were kept in climatic chamber at 42 °C and 55% relative humidity for 6 h a day between 10 : 00 h and 16 : 00 h to induce heat stress. Body weight increased significantly (p < 0.05) in CON as compared to NUS and COS. When compared within groups, scrotal width morning, scrotal width afternoon, scrotal circumference morning and scrotal circumference afternoon were significantly (p < 0.05) larger in CON while smaller in COS rams. The higher testicular length was recorded both during morning (p < 0.05) and afternoon (p < 0.01) in COS rams while the lowest in NUS rams. The highest plasma testosterone concentration was recorded in CON and lowest in COS rams. Semen volume and mass motility also differed significantly (p < 0.05) between the groups. The highest semen volume and mass motility was recorded in CON and NUS while lowest in both HES and COS rams. It can be concluded from this study that when two stressors occur simultaneously, they may have severe impact on reproductive performance of rams.

Supplementation of Slow-Release Melatonin Improves Recovery of Ovarian Cyclicity and Conception in Summer Anoestrous Buffaloes (Bubalus bubalis).

The role of melatonin as a protective neurohormone against restoring cyclicity in summer anoestrous animals in photoperiod species has gained wider acceptance. This study was designed to uncover the evidence the slow-release melatonin (MLT) has on initiation of ovarian cyclicity and conception rate (CR) in summer anoestrous buffaloes. Thus, buffaloes diagnosed as summer anoestrous (absence of overt signs of oestrus, concurrent rectal examination and radioimmunoassay for serum progesterone at 10 days interval) were grouped as untreated (Group I, sterilized corn oil, n = 8) and treated (Group II, single subcutaneous injection of MLT @18 mg/50 kg bwt in sterilized corn oil, n = 20). Animals treated and detected in oestrus were artificially inseminated (AI) followed by division into Group III (second dose of MLT on 5th day post-AI, n = 8) and Group IV (no melatonin administration, n = 10). Blood samples were collected at 4 days interval for estimation of serum MLT, progesterone and oestrogen using radioimmunoassay kit. Mean oestrous induction rate (OIR), oestrous induction interval (OII), interoestrous interval (IOI) and CR were estimated. Compared to control, concentration of melatonin was significantly (p < 0.05) higher in treated group ranging from 14.34 ± 1.72 to 412.31 ± 14.47 pg/ml whereas other two hormones did not show any concentration difference. Melatonin-administered buffaloes showed significantly (p < 0.05) higher (90%) OIR with OII of 18.06 ± 1.57 days. Results showed improvement in conception rate in buffaloes administered with post-insemination melatonin. It can be concluded from the study that slow-release melatonin supplementation restored cyclicity in summer anoestrous animals resulting in improvement in conception rate in buffaloes.

Expression of HSPs: an adaptive mechanism during long-term heat stress in goats (Capra hircus).

Menacing global rise in surface temperature compelled more focus of research over understanding heat stress response mechanism of animals and mitigation of heat stress. Twenty-four goats divided into four groups (n = 6) such as NHS (non-heat-stressed), HS (heat-stressed), HS + VC (heat-stressed administered with vitamin C), and HS + VE + Se (heat-stressed administered with vitamin E and selenium). Except NHS group, other groups were exposed to repeated heat stress (42 °C) for 6 h on 16 consecutive days. Blood samples were collected at the end of heat exposure on days 1, 6, 11, and 16. When groups compared between days, expression of all heat shock proteins (HSPs) showed a similar pattern as first peak on day 1, reached to basal level on the sixth day, and followed by second peak on day 16. The relative messenger RNA (mRNA) and protein expression of HSP 60, HSP70, and HSP90 was observed highest (P < 0.05) in HS group, followed by antioxidant-administered group on days 1 and 16, which signifies that antioxidants have dampening effect on HSP expression. HSP105/110 expression was highest (P < 0.05) on day 16. We conclude that HSP expression pattern is at least two-peak phenomenon, i.e., primary window of HSP protection on the first day followed by second window of protection on day 16. HSP60, HSP70, and HSP90 play an important role during the initial phase of heat stress acclimation whereas HSP105/110 joins this cascade at later phase. Antioxidants may possibly attenuate the HSP expression by reducing the oxidative stress.

Stimulatory effect of luteinizing hormone, insulin-like growth factor-1, and epidermal growth factor on progesterone secretion and viability of cultured bubaline luteal cells.

We evaluated the temporal (24, 48 and 72 hours) and dose-dependent (5, 10, and 100 ng/mL of LH, IGF-1, and EGF, respectively) production and secretion of progesterone (P4) in cultured luteal cells from different stages of estrous cycle as well as the expression of steroidogenic acute regulatory protein (STARD1), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), and 3ß-hydroxysteroid dehydrogenase (HSD3B), anti-apoptotic gene PCNA, and pro-apoptotic gene BAX in luteal cells of mid-luteal phase in buffalo. Samples from early luteal phase (ELP; Day 1 to 4; n = 4), mid-luteal phase (MLP; Day 5 to 10; n = 4), and late luteal phase (LLP; Day 11 to 16; n = 4) of estrous cycle were collected. Progesterone was assayed by RIA, whereas mRNA expression was determined by quantitative real-time polymerase chain reaction. Results depicted that highest dose (100 ng/mL) of LH, IGF-1, and EGF and longer duration of time brought about a (P < 0.05) rise in P4 level and expression of steroidogenic enzymes and PCNA compared with the lower level(s) and control while, all treatments (P < 0.05) inhibited BAX expression in a time dependent-manner. Analysis of interaction between stage and treatments revealed that LH treatment (P < 0.05) increased P4 production compared with IGF-1 and EGF in ELP and MLP. However in LLP, treatment with IGF-1 and EGF significantly (P < 0.05) increased P4 production compared with LH treatment. Summarizing, our study explores the steroidogenic potential of LH and growth factors across different luteal stages in buffalo, which on promoting steroidogenic enzyme expression and cell viability culminated in enhanced P4 production in luteal cells.

Temporal changes in pregnancy-associated glycoproteins across different stages of gestation in the Barbari goat.

The objective of this study was to characterize the temporal profile of pregnancy-associated glycoproteins (PAGs; isoforms 1-11) across different stages of gestation in the Barbari goat. Placentae were collected from local abattoir, classified according to crown rump length of the corresponding foetus into five groups (0-30, 31-60, 61-90, 91-120, and 121-150 days of gestation), and used for relative quantification of mRNA expression by Pfaffl method. In addition, adult female goats (pregnant, n = 7; non-pregnant, n = 5) were used to estimate weekly plasma PAG and progesterone (P4) concentrations. The relative mRNA expression of PAGs was greater (p<0.05) during 31-60 days of gestation, which correlated well with the temporal changes in plasma PAG concentrations. Relative expression of PAGs decreased steadily as gestation advanced with minimum expression observed just before parturition, except for PAG-4 and PAG-8 that showed constantly higher expression throughout pregnancy. Plasma PAG and P4 concentrations showed a distinct temporal pattern with a significant increase beginning at 2 weeks and return to basal levels by 20 weeks of gestation. However, PAG concentrations reached a peak earlier in gestation (8 weeks) than P4 (10-14 weeks). Correlation analysis indicated a strong positive association (r = 0.748, p<0.01) between plasma PAG and P4 concentrations. In conclusion, results of this study indicate a distinct temporal pattern of PAG expression and secretion during gestation in the Barbari goat. The temporal changes in PAGs and the positive association with P4 are suggestive of their role in maintenance of pregnancy and progressive foetal development.

Effect of multiple stresses (thermal, nutritional, and walking stress) on the reproductive performance of malpura ewes.

A study was conducted to assess the effect of multiple stresses (thermal, nutritional, and walking stress) on the reproductive performance of Malpura ewes. 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 twelve animals each namely, GI (n=12; Control), and GII (n=1 2; Multiple stresses). GI ewes were maintained in the shed, while GII ewes were subjected to multiple stresses (thermal, nutritional, and walking stress). The estrus % differed significantly (P< 0.05) between the groups. Estrus duration also showed similar trend to that of estrus %. Both conception and lambing rate also differed significantly (P< 0.05) between the control and multiple-stress group. In addition plasma estradiol and progesterone also showed significant difference between the groups. The study proved the detrimental effects of multiple-stresses on various reproductive parameters studied. Hence it is very pertinent to conclude that when two or more than two stressors occur simultaneously, the total impact may be severe on reproductive functions of the animals.