Influence of endometritis on the follicular dynamics, recovery, quality, gene expression, nuclear maturation and in-vitro developmental competence of oocytes in Sahiwal cattle.

Uterine infections often lead to culling of valuable animals from a herd, resulting in genetic drain. The genetic potential of problematic females could be harvested by in-vitro embryo production. The objective of this study was to evaluate the effect of clinical endometritis on follicular dynamics, recovery, quality, gene expression, nuclear maturation and in-vitro developmental competence of oocytes in Sahiwal cattle. The B-mode ultrasonography was performed to examine the uterus for the presence of pus. Based on the history and reproductive examination of cows, a total of twelve (n = 12) Sahiwal cattle were selected for the experiment: (1) healthy group (n = 6) and (2) clinical endometritis group (n = 6). The 1st ovum pick-up (OPU) was conducted on day 165 postpartum. The collected cumulus oocytes complexes (COCs) were graded into A, B, C and D grades depending on the number of layers of cumulus cells and homogeneous nature of cytoplasm. Nuclear maturation was assessed by staining the oocytes with Hoechst 33,342. The results revealed that the number of medium-sized follicle (1.3 ± 0.1 versus 0.6 ± 0.1) and total number of follicles (9.1 ± 0.7 versus 6.6 ± 0.7) were higher (p < .05) in the healthy group as compared to clinical endometritis group, respectively. Similarly, the number of oocytes recovered (5.0 ± 0.4 versus 2.8 ± 0.4), oocytes with grade A, B and C (2.9 ± 0.3 versus 1.5 ± 0.3), proportion of oocytes with grade A or B (33 ± 0.0 versus 20 ± 0.1) and nuclear maturation (68 ± 0.1 versus 55 ± 0.1) were also higher (p < .05) in the healthy group as compared to clinical endometritis group, respectively. Perhaps, cleavage rate (55.1 ± 0.1 versus 46.2 ± 0.1) and blastocyst rate (29.7 ± 0.0 versus 26.3 ± 0.1) did not differ (p > .05) between the groups. Likewise, the expression level of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in immature oocytes did not differ (p > .05) between both the groups. In conclusion, clinical endometritis has a negative effect on follicular dynamics, oocyte recovery, oocyte quality and nuclear maturation; furthermore, the developmental competence of COCs is not compromised by it.

Validation of pregnancy associated glycoproteins-based ELISA kits to determine early pregnancy status in lactating Nili-Ravi buffaloes.

The objective of the current study was to evaluate pregnancy-associated glycoproteins (PAGs) based enzyme-linked immunosorbent assay (ELISA) kits utilizing whole blood, serum, or milk samples for diagnosis of early pregnancy status in lactating Nili-Ravi buffaloes. Dairy buffaloes (n = 174) of mixed parity, 4-6 years of age, having mean (± standard deviation) days in milk 165 ± 87, and body condition score of 3.26 ± 0.34 were randomly enrolled in this study. Buffaloes were exposed to penile deviated bulls with 12 h interval for estrus detection during peak breeding season and eventually bred naturally at their respective standing estrus (day 0). Blood and milk samples were collected at days 24, 28, and 35 post-breeding to run a rapid visual pregnancy test® (RVPT) as a buffalo-side test or ELISA-based assay in the laboratory to detect early pregnancy status. Transrectal B-mode ultrasonography was performed to diagnose pregnancy at day 35 post-breeding and used as a gold standard to validate results of RVPT or ELISA-based tests. The RVPT is a visual readout test for pregnancy detection and had sensitivity (77.9 vs. 89.7 vs. 93.3%), specificity (77.9 vs. 89.7 vs. 93.3%), and accuracy (84.5 vs. 90.1 vs. 94.2%) at days 24, 28, and 35 post-breeding, respectively. The PAGs were assayed using ELISA kits in serum and had sensitivity (77.9 vs. 89.7 vs. 93.3%), specificity (84.2 vs. 87.7 vs. 93.9%), and accuracy (82.1 vs. 88.4 vs. 93.7%) at days 24, 28, and 35 post-breeding, respectively. Similarly, PAGs were also analysed using ELISA kits in milk samples and had sensitivity (77.6 vs 89.5 vs 95.0%), specificity (89.1 vs 91.9, vs 93.9%), and accuracy (85.1 vs 91.1 vs 94.3%) at days 24, 28, and 35 post-breeding, respectively. Overall, the Kappa values in this study exceeded 0.85 at day 35 post-breeding using RVPT or ELISA-based test kits in serum or milk samples, indicating a high level of agreement between PAGs detection method and gold standard for pregnancy diagnosis. The pregnancy outcomes based on ELISA-based PAGs detection at day ≥28 post-breeding had a high negative predictive value, indicating that the probability of incorrectly administering prostaglandins to pregnant buffaloes would be low if these tests were implemented on a commercial dairy herd. Taken together, it is concluded that PAGs-based determination of pregnancy using RVPT or ELISA in blood, serum, or milk samples can be used effectively for pregnancy diagnosis at ≥28 days post-breeding with more than 90% accuracy in Nili-Ravi buffaloes.

Effect of three schemes of ovum pick-up on the follicular dynamics, gene expression, and in-vitro developmental competence of oocytes in Sahiwal cattle.

This study aimed to compare the effect of three schemes of ovum pick-up (OPU) on follicular dynamics, oocytes recovery, oocytes quality, gene expression, nuclear maturation and in-vitro developmental competence of oocytes in Sahiwal cattle. Considering the follicle population, all the cows were divided equally in a 3 × 3 cross over design, and each cow received one of the three treatments: (a) twice weekly (TW; n = 6), (b) once weekly (OW; n = 6) and (c) bi-weekly OPU (BW; n = 6) in three periods, with the first OPU conducted on 4, 7 and 14 days after second dominant follicle puncture (DFP) in the TW, OW and BW OPU interval groups, respectively. The collected cumulus oocytes complexes (COCs) were graded into A, B, C and D grades depending on the number of layers of cumulus cells and homogeneous nature of cytoplasm. Nuclear maturation was assessed by staining the oocytes with Hoechst 33342. The growth rate (mm/day) of dominant follicle (DF) (F1) (0.49 ± 0.21 vs. 0.71 ± 0.26 vs. 1.30 ± 0.27) and first subordinate follicle (F2) (0.85 ± 0.27 vs. 0.71 ± 0.25 vs. 1.06 ± 0.29) did not differ (p > .05) among all the three groups. The proportion of animals bearing a corpus luteum (CL) in the BW OPU interval group (53.3%) was significantly higher (p < .05) as compared to TW (13.3%) and OW (18.3%) OPU interval groups. The number of medium-sized follicles and oocyte with grade A and B were significantly higher (p < .05) in the TW (1.16 ± 0.21 and 33.88 ± 0.03) OPU interval group as compared to the OW (0.88 ± 0.22 and 21.54 ± 0.03) and BW (0.55 ± 0.21 and 21.89 ± 0.02) OPU interval groups. However, the number of degenerated oocytes in BW (0.85 ± 0.16) OPU interval group was significantly higher (p < .05) as compared to the TW (0.16 ± 0.15) and OW (0.44 ± 0.16) OPU interval groups. Expression level of growth differentiation factor 9 in TW OPU interval group was significantly higher (p < .05) as compared to the OW and BW OPU interval groups. Likewise, expression level of bone morphogenetic protein 15 (BMP15) in the TW and BW OPU interval groups was significantly higher (p < .05) as compared to the OW OPU interval group. The nuclear maturation rate was significantly higher in the TW (63.64 ± 0.07) and BW (59.26 ± 0.08) OPU groups as compared to OW (51.43 ± 0.06) OPU interval group. However, the cleavage rate (59.30 ± 0.06 vs. 44.29 ± 0.06 vs. 56.67 ± 0.06) did not differ (p > .05) among the three groups. Whereas, the blastocyst rate tended to be higher (p = .06) in the TW (29.07 ± 0.05) and BW (28.33 ± 0.04) OPU interval groups as compared to OW (18.57 ± 0.05) OPU interval group. Taken together, it can be concluded that TW OPU interval scheme enhances the medium-sized follicles resulting in good quality oocytes, regulates the oocyte-derived paracrine factors, leading to higher nuclear maturation rates and improved embryonic development in-vitro.

Development of nutritious probiotic sportsman drink.

Whey plays an important role in the sports nutrition because of high quality proteins and essential amino acid profile. Nine formulations of sportsman drinks were made using Cheddar, Mozzarella and Paneer whey with normal as well as additional fermentation. The developed sportsman drinks were evaluated for physico-chemical analyses, amino acid profile, viscosity and total plate count along with sensory response during two month storage. Drink having Cheddar whey (T4) with additional fermentation was better in terms of quality and nutrition. Furthermore, amino acid profile considered it a complete and balanced source of essential and non-essential amino acids. Amongst essential amino acids, highest values was recorded for branched chain amino acids like leucine (73.16±3.09) followed by lysine (61.56±0.61) and valine (44.13±1.86)mg/g protein. The dietary significance of sportsman drink can be enhanced through additional fermentation using Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophillus.

Synthesis, computational studies, tyrosinase inhibitory kinetics and antimelanogenic activity of hydroxy substituted 2-[(4-acetylphenyl)amino]-2-oxoethyl derivatives.

The over expression of melanogenic enzymes like tyrosinase caused many hyperpigmentaion disorders. The present work describes the synthesis of hydroxy substituted 2-[(4-acetylphenyl)amino]-2-oxoethyl derivatives 3a-e and 5a-e as antimelanogenic agents. The tyrosinase inhibitory activity of synthesized derivatives 3a-e and 5a-e was determined and it was found that derivative 5c possesses excellent activity with IC50 = 0.0089 µM compared to standard kojic acid (IC50 = 16.69 µM). The presence of hydroxyl groups at the ortho and the para position of cinnamic acid phenyl ring in compound 5c plays a vital role in tyrosinase inhibitory activity. The compound 5d also exhibited good activity (IC50 = 8.26 µM) compared to standard kojic acid. The enzyme inhibitory kinetics results showed that compound 5c is a competitive inhibitor while 5d is a mixed-type inhibitor. The mode of binding for compounds 5c and 5d with tyrosinase enzyme was also assessed and it was found that both derivatives irreversibly bind with target enzyme. The molecular docking and molecular dynamic simulation studies were also performed to find the position of attachment of synthesized compounds at tyrosinase enzyme (PDB ID 2Y9X). The results showed that all of the synthesized compounds bind well with the active binding sites and most potent derivative 5c formed stable complex with target protein. The cytotoxicity results showed that compound 5c is safe at a dose of 12 µg/mL against murine melanoma (B16F10) cells. The same dose of 5c was selected to determine antimelanogenic activity; the results showed that it produced antimelenogenic effects in murine melanoma (B16F10) cells. Based on our investigations, it was proposed that compound 5c may serve as a lead structure to design more potent antimelanogenic agents.

Effect of breeding method and season on pregnancy rate and embryonic and fetal losses in lactating Nili-Ravi buffaloes.

The aim of this study was to determine the effect of breeding method and season on pregnancy rate and cumulative embryonic and fetal losses in Nili-Ravi buffalo. Estrus detection was performed twice a day by teaser buffalo bull for 1 hour each. A 2 × 2 factorial design was used to address the breeding method and season. Buffaloes (n = 130) exhibiting estrus were randomly assigned to be bred either in peak breeding season (PBS; n = 80) or low breeding season (LBS; n = 50). Within each season, buffaloes were divided to receive either natural service (NS; n = 65) or artificial insemination (AI; n = 65). NS buffaloes, in estrus, were allowed to remain with the bull until mating. AI was achieved, using frozen thawed semen of bull of known fertility. PBS comprised of September to December and LBS were from May to July. Serial ultrasonography was performed on days 30, 45, 60, and 90 after breeding (day 0) to monitor pregnancy rate and embryonic and fetal losses. The pregnancy rate on day 30 after breeding was higher in NS as compared to AI group (63 vs. 43%; P < 0.05) during PBS while it did not differ (48 vs. 32%; P > 0.05) in LBS. The cumulative embryonic and fetal losses between days 31 and 90 were significantly lower in PBS than LBS (33 vs. 60%; P < 0.05), ignoring breeding method. Pregnancy rates were better with NS in PBS, and cumulative embryonic fetal losses were higher in LBS in Nili-Ravi buffalo.

Effectiveness of clobazam on perception, creativity, intelligence, selective and visual memory.

Clobazam belongs to benzodiazepine class and is preferably used against anti-epileptic disorders. However, when used in reduced doses, its ability for improving cognitive functions becomes explicitly evident. This study objectively undertook the task of using the reduced doses of clobazam for proving potentials effects on cognitive functions. The drug, clobazam was administered in "active group" which contained 15 young healthy volunteers. The "placebo group" also entailed 15 subjects and each was administered with placebo drug. The controlled group? also included 15 subjects. All these 45 young healthy subjects were subjected to tests for perceptual learning, creativity, selective memory, visual memory and intelligence. Results clearly demonstrated significant impact of clobazam at the dose of 5mg/day on perceptual learning (P=0.0380), creativity (P=0.0787), memory function (P=0.4920), visual memory (P=0.4816) and intelligence of the subject (P=0.4920). The outcomes highlighted in the studies reviled the positive effects of clobazam when used at reduced doses.

Effect of melatonin on regulation of apoptosis and steroidogenesis in cultured buffalo granulosa cells.

This study was aimed to address melatonin receptor expression, mRNA level of hypothalamus and hypophysis hormone receptors (GnRHR, FSHR, and LHR), steroidogenesis, cell cycle, apoptosis, and their regulatory factors after addition of melatonin for 24 hr in cultured buffalo granulosa cells (GCs). The results revealed that direct addition of different concentrations of melatonin (100 pM, 1 nM, and 100 nM) resulted in significant upregulation (p < 0.05) of mRNA level of melatonin receptor 1a (MT1) without affecting melatonin receptor 1b (MT2). Melatonin treatment significantly downregulated (p < 0.05) mRNA level of FSH and GnRH receptors, whereas 100 nM dose of melatonin significantly increased mRNA level of LH receptor. Treatment with 100 nM of melatonin significantly decreased the basal progesterone production with significant decrease (p < 0.05) in mRNA levels of StAR and p450ssc, and lower mRNA level of genes (Insig1, Lipe, and Scrab1) that affect cholesterol availability. Melatonin supplementation suppressed apoptosis (100 nM, p < 0.05) and enhanced G2/M phase (1 nM, 100 nM, p < 0.05) of cell cycle progression which was further corroborated by decrease in protein expression of caspase-3, p21, and p27 and increase in bcl2. Our results demonstrate that melatonin regulates gonadotrophin receptors and ovarian steroidogenesis through MT1. Furthermore, the notion of its incorporation in apoptosis and proliferation of buffalo GCs extends its role in buffalo ovaries.

Activity and Anti-Aflatoxigenic Effect of Indigenously Characterized Probiotic Lactobacilli against Aspergillus flavus-A Common Poultry Feed Contaminant.

Aflatoxin contamination in human food and animal feed is a threat to public safety. Aflatoxin B1 (AFB1) can be especially damaging to poultry production and consequently economic development of Pakistan. The present study assessed the in vitro binding of AFB1 by indigenously characterized probiotic lactobacilli. Six isolates (Lactobacillus gallinarum PDP 10, Lactobacillus reuetri FYP 38, Lactobacillus fermentum PDP 24, Lactobacillus gallinarum PL 53, Lactobacillus paracasei PL 120, and Lactobacillus gallinarum PL 149) were tested for activity against toxigenic Aspergillus flavus W-7.1 (AFB1 producer) by well diffusion assay. Only three isolates (PL 53, PL 120, and PL 149) had activity against A. flavus W-7.1. The ameliorative effect of these probiotic isolates on AFB1 production was determined by co-culturing fungus with lactobacilli for 12 days, followed by aflatoxin quantification by high-performance liquid chromatography. In vitro AFB1 binding capacities of lactobacilli were determined by their incubation with a standard amount of AFB1 in phosphate buffer saline at 37 °C for 2 h. AFB1 binding capacities of isolates ranged from 28-65%. Four isolates (PDP 10, PDP 24, PL 120, and PL 149) also ceased aflatoxin production completely, whereas PL 53 showed 55% reduction in AFB1 production as compared to control. The present study demonstrated Lactobacillus gallinarum PL 149 to be an effective candidate AFB1 binding agent against Aspergillus flavus. These findings further support the binding ability of lactic acid bacteria for dietary contaminants.

Equine chorionic gonadotropin (eCG) enhances reproductive responses in CIDR-EB treated lactating anovular Nili-Ravi buffalo during the breeding season.

The objective of this study was to evaluate the effect of equine chorionic gonadotropin (eCG) on the estrous response, ovulatory follicle development, ovulation rate, corpus luteum (CL) diameter, concentrations of progesterone (P4) and pregnancy per AI (P/AI) in CIDR-EB treated lactating anovular Nili-Ravi buffalo during the breeding season. Buffalo (n = 87), 60 to 200 Days postpartum were treated with CIDR at a random stage of the estrous cycle (Day 0) and assigned to two groups; with eCG (+eCG; n = 44), or without eCG (-eCG; n = 43). In both groups, each animal received 2 and 1 mg estradiol benzoate (EB) on Day 0 and then on Day 8, respectively. On Day 6, eCG and PGF2α were administered. On Day 7, CIDR were removed and FTAI was performed at 48 and 60 h later and pregnancy was diagnosed 35 Days post AI. The results revealed that eCG treatment increased (P <  0.05) the CL diameter on 15, 18, and 21 after the ovulation that resulted from imposing the estrous synchronization protocol than when eCG was not administered. Similarly, treatment with eCG resulted in an increased (P <  0.05) mean concentration of P4 as compared with animals not treated with eCG on Days 18 and 21 after the ovulation that occurred subsequent to imposing the estrous synchronization protocol. The ovulation rate was greater in eCG-treated buffalo as compared with those not treated with eCG (93% compared with 74%, respectively; P < 0.05). The P/AI was greater in buffalo treated with eCG than those not treated with eCG (56% compared with 31%, respectively; P < 0.05). In conclusion, the addition of eCG along with EB and P4 based treatments enhanced the estrous response, follicular and luteal function, ovulation rate, concentrations of P4, and P/AI in lactating anovular Nili-Ravi buffalo.

Presynchronization of lactating dairy cows with PGF2α and GnRH simultaneously, 7 days before Ovsynch have similar outcomes compared to G6G.

The overarching objective of this study was to develop an alternative strategy for first and greater services that will improve fertility in lactating dairy cows for dairy operations limited by labor or other logistical constraints that make it difficult to use Presynch-11, G6G, or Double-Ovsynch. Our overall hypothesis was that simplification of a Presynch program through the combination of PGF2α and GnRH on the same day (PG + G), 7 days before the first GnRH of Ovsynch, would allow for similar ovulation and luteolysis rate and pregnancies per AI (P/AI) compared with G6G. Lactating dairy cows 58 to 64 days in milk (first service; n = 114), and cows diagnosed not pregnant 39 days after previous AI (second + service; n = 122) were blocked by parity and service and randomly assigned to control or PG + G. Control cows received G6G (n = 116) that consisted of PGF2α, 2-day GnRH, 6-day GnRH, 7-day PGF2α, 56-hour GnRH, and 16-hour AI. Treated cows (PG + G; n = 121) received PGF2α and GnRH, 7-day GnRH, 7-day PGF2α, 56-hour GnRH, and 16-hour AI. All cows received a second PGF2α 24 hours after the PGF2α of Ovsynch. First service cows received AI between 76 and 82 days in milk and second + service received AI 57 days after previous AI. Pregnancies/AI (n = 230) were similar in controls compared with treated cows on Day 35 (57 vs. 50%; P = 0.27) and Day 49 (54 vs. 47%; P = 0.33), respectively. Percent of cows ovulating after GnRH of the presynchronization was greater (P = 0.002) for controls vs. treated (80 vs. 58%); however, ovulation after first GnRH of Ovsynch was similar (67 vs. 68%; P = 0.86). Serum concentrations of progesterone were similar (P = 0.78) at the time of first GnRH of Ovsynch for control and treated cows (2.22 vs. 2.14 ng/mL). However, serum progesterone at the time of PGF2α of Ovsynch was greater (P = 0.002) for control cows compared with treated cows (5.75 vs. 4.64 ng/mL). In summary, administering both PGF2α and GnRH on the same day, 7 days before the start of Ovsynch, appears to be a simple alternative that results in acceptable P/AI but potentially less progesterone during the growth of the ovulatory follicle.

Effect of oestradiol benzoate on oestrus intensity and pregnancy rate in CIDR treated anoestrus nulliparous and multiparous buffalo.

The objective of the present study was to examine if administration of oestradiol benzoate (OEB) after removal of control internal drug releasing device (CIDR) could enhance oestrous intensity and pregnancy rate in anovular nulliparous and multiparous Nili-Ravi buffalo. For this, a total of 298 nulliparous (n=91) and multiparous (n=207) buffaloes received a CIDR on Day 0, and were administered PGF2α on Day 6 followed by removal of CIDR on Day 7. At Day 8, OEB was administered in approximately half of nulliparous (n=45/91) and multiparous (n=100/207) buffalo. All animals were fixed time inseminated 48 and 60h after CIDR removal, respectively. The results showed that administration of OEB but not the parity, improved oestrous intensity (3.15±0.05 vs 2.99±0.05; P=0.0026) compared to those not received OEB, respectively. However, OEB did not affect (46.2 vs 44.1; P=0.8) pregnancy per AI (P/AI). In addition, P/AI was greater (50.7 vs 39.6; P=0.036) in multiparous compared to nulliparous buffalo, respectively. The oestrous intensity (P=0.025) and response to OEB (P=0.0002) was greater in buffalo having a greater body condition (>3.0). Though, non significant, timing of ovulation was more synchronous (62.9±1.8 vs 72.4±3.6h; P>0.05) and ovulation rate was greater (91% vs 64%; P>0.05) in buffalo after OEB administration. It is concluded that administration of OEB in conjunction with the CIDR improves oestrous intensity without affecting P/AI in nulliparous and multiparous anovular buffalo.