Effect of dietary fish oil replacement with palm oil on growth performance,hematology and liver anti‐oxidative enzymes of juvenile Japanese flounder Paralichthys olivaceus (Temminck & Schlegel, 1846)

A 60‐day feeding trial was conducted to evaluate the effects of dietary palm oil supplements on growth performances, hematology, liver anti‐oxidative enzymes and air exposure resistance of Japanese flounder, Paralichthys olivaceus (initial weights 2.56 ± 0.01 g). Five diets were tested wherein the dietary fish oil was replaced by palm oil at: 0% (Control), 20% (20%), 40% (40%), 50% (50%) and 60% (60%). After the feeding trial, the 20% dietary palm oil was shown to provide similar growth rates and feed efficiency with no negative effects compared to the control group (P > 0.05). Significantly lower growth rates and feed utilization were found in fish fed higher than 40% palm oil in the diet (P < 0.05). Except for total serum protein, the blood parameters, liver anti‐oxidative enzymes, stress resistance and proximate compositions of Japanese flounder were not altered, even with dietary palm oil up to 60% of the lipid source (P > 0.05). According to the present results, palm oil is a valuable lipid source substitute in Japanese flounder diets; around 20–40% fish oil can be replaced with palm oil with no negative effects.     

Effects of birth weight and maternal dietary fat source on the fatty acid profile of piglet tissue

This study aimed to investigate the effects and possible interactions of birth weight and n-3 polyunsaturated fatty acid (PUFA) supplementation of the maternal diet on the fatty acid status of different tissues of newborn piglets. These effects are of interest as both parameters have been associated with pre-weaning mortality. Sows were fed a palm oil diet or a diet containing 1% linseed, echium or fish oil from day 73 of gestation. As fish oil becomes a scarce resource, linseed and echium oil were supplemented as sustainable alternatives, adding precursor fatty acids for DHA to the diet. At birth, the lightest and heaviest male piglet per litter were killed and samples from liver, brain and muscle were taken for fatty acid analysis. Piglets that died pre-weaning had lower birth weights than piglets surviving lactation (1.27±0.04 v. 1.55±0.02 kg; P<0.001), but no effect of diet on mortality was found. Lower DHA concentrations were observed in the brain of the lighter piglets compared with their heavier littermates (9.46±0.05 v. 9.63±0.04 g DHA/100 g fatty acids; P=0.008), suggesting that the higher incidence of pre-weaning mortality in low birth weight piglets may be related to their lower brain DHA status. Adding n-3 PUFA to the sow diet could not significantly reduce this difference in DHA status, although numerically the difference in the brain DHA concentration between the piglet weight groups was smaller when fish oil was included in the sow diet. Independent of birth weight, echium or linseed oil in the sow diet increased the DHA concentration of the piglet tissues to the same extent, but the concentrations were not as high as when fish oil was fed.     

L-carnitine is a survival factor for chilled storage of rooster semen for a long time

Rooster sperm is sensitive to cooling, which restricts procedures to store sperms for extended periods of time for artificial insemination of commercial flocks. This study was conducted to evaluate the suitability of adding L-carnitine (LC) to chilled-storage of rooster sperm and its effects on sperm quality parameters and its fertility potential during storage at 5 °C. Pooled semen from roosters were divided into six equal aliquots and diluted with media supplemented with different concentrations of LC (0, 0.5, 1, 2, 4 and 8 mM LC). Diluted semen samples were cooled to 5 °C and stored over 48 h. Motility, viability, membrane functionality, lipid peroxidation and mitochondria activity of the sperm were assessed at 0, 24 and 48 h of storage. Moreover, fertility potential of chilled stored sperm was considered at 24 h of storage. While sperm quality was not affected by LC at the beginning of storage (0 h), supplementation of extender with 1 and 2 mM of LC significantly improved the percentage of sperm motility, viability, membrane integrity and mitochondria activity at 24 h and 48 h compared to other groups. Lipid peroxidation was significantly reduced in sperm samples diluted with 1 and 2 mM LC at 24 h (2.15 ± 0.52 nmol/ml and 2.21 ± 0.52 nmol/ml) and 48 h (3.42 ± 0.49 nmol/ml and 3.38 ± 0.49 nmol/ml) compared to other groups. Furthermore, fertility rates during artificial insemination using sperms cooled for 24 h in the presence of 1 and 2 mM LC were significantly higher (78%) than in the control group (64%). These findings suggest that optimum doses of LC could protect rooster sperm against cool storage-induced functional and structural damages.     

Increased quality of in natura and cryopreserved semen of water buffaloes supplemented with saturated and unsaturated fatty acids from the palm oil industry

Ruminant energy supplementation with vegetable oils or fats has been standing out worldwide and oil palm processing has been receiving growing interest. This study assessed the effect of supplementation with saturated and unsaturated fatty acids from the palm oil industry on the lipid profile of seminal plasma and of the sperm membrane, as well as on the morphological and functional characteristics of raw and cryopreserved buffalo semen. Twelve purebred Murrah bulls (Bubalus bubalis) were assigned to the experimental groups and fed diets for 120 days with no added lipids (CONT, four bulls), or with an extra amount of 3% lipids from crude palm oil (PALM, four bulls), or from palm oil deodorizer distillate (PODD, four bulls). Semen was collected and cryopreserved every 15 days. The lipid composition of membranes and semen quality were determined after collections. Lipid supplementation did not impact feed intake (P>0.05). Diet enrichment with PALM increased the linoleic acid (C18:2,ω6) in seminal plasma. Lipid supplementation did not increase the polyunsaturated fatty acids in the sperm membrane composition, but significantly increased the lignoceric acid (C24:0). Cryopreserved semen of the supplemented bulls presented higher progressive motility (60.2 vs. 67.9 vs. 65.2%; P<0.05) and sperm viability detected by eosin-nigrosin staining (61.1 vs. 69.4 vs. 67.8%; P<0.05). Palm oil reduced major sperm defects in both raw (12.2 vs. 9.3 vs. 13.2%; P<0.0001) and cryopreserved semen (12.4 vs. 9.4 vs. 11.2%; P<0.0001). The lipids added to the diet did not impact the population of spermatozoa with intact plasma and acrosomal membranes (PI-/PSA-), but significantly increased the percentage of spermatozoa with high mitochondrial potential (25.6 vs. 31.5 vs. 32.0%; P=0.008). The results suggest that lipid supplementation based on crude palm oil or palm oil deodorizer distillate can be safely used to feed buffalo bulls and may increase sperm attributes related to male fertility.     

Effect of bioactive peptide on ram semen cryopreservation

This present study investigated the effect of bioactive peptide (BAPT) (BAPT) on the quality of ram semen during cryopreservation. Ram ejaculates were extended with Tris buffer supplemented with no antioxidants (as control group), 20 μg/mL BAPT (as BAPT20 group), 40 μg/mL BAPT (as BAPT40 group) and 60 μg/mL BAPT (as BAPT60 group). After cryopreservation, sperm quality including motility, vitality, the percentage of hypoosmotic swelling test (HOST)-positive spermatozoa and the percentage of intact acrosomes was assessed. Furthermore, the malondialdehyde (MDA) in seminal plasma and spermatozoa were analyzed, followed by the measurement of superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GSH-Px) levels in seminal plasma. After in vitro fertilization, the embryonic cleavage rates and development rates of different groups were analyzed to compare the developmental abilities of spermatozoa. The results showed that the post-thaw sperm motility was significantly higher in the BAPT60 group compared to those in the BAPT20, BAPT40 and control groups (P < 0.05). The percentage of live sperms significantly increased from 48.12 ± 2.35% for the BAPT20 group, 55.43 ± 2.16% for the BAPT40 group to 57.53 ± 3.15% for the BAPT60 group. The percentage of HOST-positive spermatozoa was significantly higher in the BAPT60 group than those in BAPT20, BAPT40 and control groups (P < 0.05). The MDA levels in seminal plasma and spermatozoa were significantly reduced with BAPT supplement (P < 0.05). Additionally, the SOD, CAT and GSH-Px levels in the BAPT experimental groups were significantly higher than those of the control group, which further indicated that BAPT significantly inhibit the reactive oxygen species (ROS) production during the cryopreservation of ram semen. Furthermore, the embryonic cleavage rates and development rates of the BAPT40 and BAPT60 groups were significantly increased in comparison with the BAPT20 and control groups (P < 0.05).In conclusion, BAPT improved the ram sperm quality via inhibiting the ROS production during cryopreservation, and could be applied as a promising supplement for ram semen cryopreservation.     

Influence of dietary polyunsaturated fatty acids on renal & aortic prostaglandin synthesis in 1 kidney 1 clip goldblatt hypertensive rats

To study the influence of dietary modification on prostaglandin synthesis and on blood pressure regulation, the effects of dietary enrichment with linolenic or linoleic acid was compared with standard rat chow in 3 groups of 13 rats before and after renal artery constriction and contralateral nephrectomy. Before renal artery constriction 4 weeks supplementation with 40 en% linseed oil (53% linolenic acid) increased renal linolenic acid, decreased arachidonic acid, and suppressed synthesis of 6-keto-PGF_1α and PGE₂ by renal homogenates (33% and 38% respectively, p<0.01) compared with standard diet. Rats fed on 40 en % sunflower seed oil (63% linoleic acid) increased renal prostaglandin synthesis (p<0.05) compared with linseed oil, but not compared with standard diet. Seven weeks after renal artery constriction renal and aortic 6-keto-PGF_1α and PGE₂ were suppressed 30% to 50% (p<0.05) by linseed oil supplements compared with sunflower seed oil and standard diets. In the sunflower seed oil group aortic 6-keto-PGF_1α correlated (r = 0.75, p<0.02) with final systolic blood pressure. Final systolic blood pressures were similar in linseed oil (152.9 mmHg ± se 3.3, sunflower oil (155.1 ± se 6.6) and standard diet group (159.0 ±se 4.2). Thus dietary linseed oil suppressed renal and aortic prostaglandin synthesis but did not accentuate renal hypertension, and linoleic acid supplementation did not protect against 1 kidney 1 clip renal hypertension.     

Effects of different nitrogen rich substrates and their combination to the yield performance of oyster mushroom (<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>)

This study was conducted to assess the effect of nitrogen (N) variations from lignocellulosic substrates of empty fruit bunch (EFB), palm oil frond (POF), palm oil trunks (POT), A.mangium bark (AB) and Cogongrass (IC), and combinations among them using biological supplements (POF and POT) for suitability as a substrate of P. ostreatus. Three different sets of substrates were evaluated in response to different parameters of P. ostreatus including spawn running, primordial initiation, fruit body yield and biological efficiency (BE). The supplementation of POT and POF generally revealed significant differences on the crop performance of P. ostreatus compared to the un-supplemented substrate. POT demonstrated reduced production time, while POF was the best supplement to promote fresh weight and BE. The most rapid spawn running took place within 18.6 ± 0.9 days, while the primordial initiation was observed within 23.2 ± 0.5 days in the combination of IC + POT. The most suitable substrate for higher mushroom weight and BE was IC + POF (19.6 ± 1.3 g; 70.5 ± 5.5%). In addition, the interval between primordial formations was found to be fastest in the un-supplemented EFB and the combination of EFB + POF, which supported a shortened production time. Therefore, IC and EFB appeared to be the better alternative materials for growing P. ostreatus, either with or without any supplementation.     

Effects of label-dose permethrin administration in yearling beef cattle: I. Bull reproductive function and testicular histopathology

Pyrethroid administration to a wide variety of laboratory animals has been shown to cause detrimental effects on male fertility, including sperm quality, by means of endocrine disruption. The objective of this experiment was to study the effects of a commercial, permethrin-containing pour-on product on reproductive variables and testicular histopathology of yearling beef bulls. Black Angus bulls (n = 60; aged 369 ± 17 days; 511 ± 33 kg; 6.2 ± 0.5 body condition scores) were assigned to either (1) saline control (CON) or (2) permethrin pour-on administered at label dose (PYR). Blood samples were collected, and industry standard breeding soundness examinations (BSE), via electroejaculation, were performed on all bulls at 5 days before and 14 days after treatment. Progressive sperm motility and eosin-nigrosin–stained sperm were analyzed using high-power phase-contrast microscopy. Plasma testosterone concentrations were analyzed via radioimmunoassay. Bulls were slaughtered at 34 days, and one testicle per bull was randomly collected for histologic examination. Change in sperm motility between BSEs was not different because of treatment; sperm morphology however improved across treatments, but PYR bulls had less improvement in percent of head (P < 0.001) sperm abnormalities compared to CON, resulting in less improvement of primary abnormalities (P = 0.04). Nonetheless, morphological differences did not change the overall outcome for satisfactory breeder status. Change in testosterone concentration did not differ because of treatment. Histopathologic examination identified that testicular degeneration and tubule diameter did not differ as a result of treatment. It should be noted, however, that degeneration score (higher score having more degeneration) was positively correlated with primary abnormalities (P < 0.01; r = 0.35) and negatively correlated with normal sperm cells (P < 0.001; r = −0.43). In summary, these data indicate that a single use of permethrin at label dose in yearling Angus bulls results in minimal detrimental effects on sperm morphology but not to a degree that impacts the ability of bulls to pass a standard BSE.     

Effect of storage of domestic cat (Felis catus) epididymides at 5 °C on sperm quality and cryopreservation

Postmortem sperm recovery from the epididymides may constitute a powerful tool for the conservation of valuable genetic material. The domestic cat (Felis catus) is a good model for wild felids and, using this model, we have explored the effect of epididymides storage time on sperm motility and percentage of intact acrosomes upon sperm recovery and after cryopreservation. We also examined the effect of time of sperm equilibration with glycerol before freezing on sperm motility and the percentage of intact acrosomes. Motility varied between sperm recovered from epididymides that were stored for different times. Significant differences were seen in the sperm motility index (SMI) before freezing (55.91 ± 2.02, 48.21 ± 1.47, and 43.03 ± 1.32) and after thawing (51.81 ± 3.02, 41.90 ± 2.14, and 42.35 ± 1.95) of sperm recovered from epididymides stored for 0, 48, or 72 h, respectively. The percentage of intact acrosomes did not vary significantly with storage time (average 60.33 ± 1.38% before and 52.50 ± 1.91% after freezing, respectively). The percentage of normal sperm after different storage times did not differ (average 19.22 ± 1.25% normal sperm after recovery). When epididymides were stored for 72 h, time of sperm equilibration with glycerol (30 vs. 120 min) resulted in significant differences in both motility (SMI = 39.17 ± 2.76 and 45.00 ± 2.65, respectively) and the percentage of intact acrosomes (45.76 ± 4.91% and 60.67 ± 3.64%, respectively) after thawing. In conclusion, best results are achieved when sperm are recovered from epididymides within 24 h of cool storage and when they are equilibrated with glycerol during 120 min before freezing. The current results should be useful in the further development of techniques for the rescue and cryostorage of epididymal spermatozoa of endangered felids.     

Extinction thresholds and negative responses of Afrotropical ant-following birds to forest cover loss in oil palm and agroforestry landscapes

Afrotropical ant-following birds are vulnerable to forest loss and disturbance, but critical habitat thresholds regarding their abundance and species richness in human-dominated landscapes, including industrial oil palm plantations, have never been assessed. We measured forest cover through Landsat imagery and recorded species richness and relative abundance of 20 ant-following birds in 48 plots of 1-km², covering three landscapes of Southwest Cameroon: Korup National Park, smallholder agroforestry areas (with farms embedded in forest), and an industrial oil palm plantation. We evaluated differences in encounter frequency and species richness among landscapes, and the presence of critical thresholds through enhanced adaptive regression through hinges. All species were detected in Korup National Park and the agroforestry landscape, which had similar forest cover (>85%). Only nine species were found in the oil palm plantation (forest cover = 10.3 ± 3.3%). At the 1-km² scale, the number of species and bird encounters were comparable in agroforests and the protected area: mean species richness ranged from 12.2 ± 0.6 in the park and 12.2 ± 0.6 in the agroforestry matrix to 1.0 ± 0.4 in the industrial oil palm plantation; whereas encounters decreased from 34.4 ± 3.2 to 26.1 ± 2.9 and 1.3 ± 0.4, respectively. Bird encounters decreased linearly with decreasing forest cover, down to an extinction threshold identified at 24% forest cover. Species richness declined linearly by ca. one species per 7.4% forest cover lost. We identified an extinction threshold at 52% forest cover for the most sensitive species (Criniger chloronotus, Dicrurus atripennis, and Neocossyphus poensis). Our results show that substantial proportions of forests are required to sustain complete ant-following bird assemblages in Afrotropical landscapes and confirm the high sensitivity of this bird guild to deforestation after industrial oil palm development. Securing both forest biodiversity and food production in an Afrotropical production landscape may be best attained through a combination of protected areas and wildlife-friendly agroforestry.     

Royal jelly supplemented soybean lecithin-based extenders improve post-thaw quality and incubation resilience of goat spermatozoa

The aim of the present study was to evaluate different concentrations of royal jelly (RJ) supplemented extenders for post-thaw quality and incubation resilience of goat spermatozoa. Semen samples were collected from five goats. Pooled semen were diluted with soybean lecithin-based extender without RJ (control) or supplemented with different concentrations (0.25, 0.5 and 0.75%) of RJ (RJ0.25, RJ0.5, RJ0.75 respectively), at a final concentration of 150 × 10⁶ spermatozoon/mL. Semen samples were assessed for sperm motility, plasma membrane integrity using hypoosmotic swelling test (HOST) damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).The addition of RJ (0.5%, 0.75%) led to higher percentages of subjective motilities (55.33 ± 2.29%, 57.67 ± 2.58%) compared to control and RJ0.25 groups (49.00 ± 2,80%, 51.67 ± 3.09%) (P < 0.05) following the freeze–thawing process. RJ0.5 and RJ0.75 groups had higher plasma membrane functional integrities (66.40 ± 1.34%, 68.20 ± 2.05%) and lower defected acrosome rates (24.60 ± 3.36%, 23.80 ± 2.27%) compared to the other groups (P < 0.05). DNA damaged spermatozoa in all groups were not significant (P > 0.05).In the end of incubation, motility and HOST rates of RJ0.5 (14.00 ± 3.87%, 31.20 ± 3.70%) and RJ0.75 (15.00 ± 3.27%, 29.20 ± 2.59%) groups were higher than control (8.00 ± 2.54%, 18.20 ± 3.11%) and RJ0.25 (9.00 ± 2.07%, 20.60 ± 2.88%) groups (P < 0.05). Also defected acrosome and DNA fragmation rates of RJ0.5 (32.20 ± 1.30%, 5.4 ± 0.55%) and RJ0.75 (29.20 ± 1.30%, 5.80 ± 0.45%) groups were significantly lower than control (38.80 ± 0.84%, 7.40 ± 1.34%) and RJ0.25 (39.80 ± 2.05%, 7.00 ± 1.58) groups. This study shows that RJ supplemented extenders have beneficial effect on goat sperm parameters at 0 h and 6 h of incubation.     

Phytosterols accumulation in the seeds of Linum usitatissimum L.

A comparative study was performed to determine the free sterols content and composition during the development of three varieties of linseed (H52, O116 and P129). Seed samples were collected at regular intervals from 7 to 60 days after flowering (DAF). Ten compounds were identified: cholesterol, campesterol, brassicasterol, stigmasterol, β-sitosterol, Δ5-avenasterol, cycloartenol; 24-methylene cycloartanol, obtusifoliol, citrostadienol. The maximum level of 4-desmethylsterols (1515 mg/100 g oil) was reached at 7 DAF in P129 variety. H52 had the highest level of 4-4 dimethylsterols (355 mg/100 g oil) at 28 DAF. The greatest amount of 4-monomethylsterols (35 mg/100 g oil) was detected in H52 at 14 DAF. During linseed development, β sitosterol (830 mg/100 g oil) was the major 4-desmethylsterols, followed by campesterol (564 mg/100 g oil) and stigmasterol (265 mg/100 g oil). Some of these compounds followed nearly the same accumulation pattern during linseed maturation.     

Comparison of methods for assessing integrity of equine sperm membranes

Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Bland-Altman methodology. Eosin-nigrosin staining yielded higher (2%) overall mean values for SMI than did flow cytometry. Flow cytometry yielded higher (6%) overall mean values for SMI than did the automated cell counter. As percentage of membrane-damaged sperm increased, agreement of SMI measurement between methods decreased. When semen contained 50-79% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = −26.9 to 24.3%; i.e., a 51.2% span) than for SMI determined by flow cytometry and the automated cell counter (range = −3.1 to 17.0%; 20.1% span). When sperm populations contained <50% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = −35.9 to 19.0%; 54.9% span) than for SMI determined by flow cytometry and the automated cell counter (range = −11.6 to 28.7%; 40.3% span). We concluded that eosin-nigrosin staining assessments of percent membrane-intact sperm agreed less with flow cytometry when <80% of sperm had intact membranes, whereas automated cell counter assessments of percent membrane-intact sperm agreed less with flow cytometry when <30% of sperm had intact membranes.     

Transplantation of Neural Stem Cells Overexpressing Cardiotrophin-1 Inhibits Sprouting of Hippocampal Mossy Fiber in a Rat Model of Status Epilepticus

In this study, we studied the effects of hippocampal transplantation of neural stem cells (NSCs) overexpressing cardiotrophin 1 (CT1) on hippocampal mossy fiber sprouting (MFS) in a rat model of status epilepticus (SE). SE rats (lithium–pilocarpine model) were randomized into four study groups (18 rats per group): CT1-NSCs group, NSCs group, SE control group, and normal control group. Six rats were randomly chosen from each group at 1, 4, and 8 weeks after transplantation. MFS in hippocampal dentate gyrus was scored (Timm staining) at these time points. The MFS scores were as follows: CT1-NSCs 0.77 ± 0.04, 2.48 ± 0.89, and 2.39 ± 0.82 (1, 4, and 8 weeks after transplantation, respectively); NSCs 1.12 ± 0.62, 3.17 ± 0.64, and 3.88 ± 0.51; SE control 1.32 ± 0.35, 3.28 ± 0.75, and 4.32 ± 1.55; and normal control 0.37 ± 0.06, 0.34 ± 0.07, and 0.43 ± 0.04. Compared to SE control group and NSCs group, the scores of MFS in CT1-NSCs group were significantly lower (P < 0.05). In conclusion, transplantation with NSCs overexpressing CT1 inhibits hippocampal MFS and facilitates reduction of recurrent seizures.     

Successful sperm cryopreservation of the brown-marbled grouper,Epinephelus fuscoguttatus using propylene glycol as cryoprotectant

This study developed the cryopreservation of brown-marbled grouper spermatozoa for practical application. We examined 32 cryodiluents, developed from four types of cryoprotectants [propylene glycol (PG), dimethyl-sulphoxide (Me₂SO), dimethyl-acetamide (DMA) and ethylene glycol (EG)] at four concentrations of 5, 10, 15 and 20% in combination with two extenders [Fetal bovine serum (FBS) and artificial seminal plasma (ASP). Cooling rates were examined by adjusting the height of straws (2.5–12.5 cm) from the liquid nitrogen (LN) vapor and cooled for 5 min before immersion into LN. DNA laddering was used to detect DNA damage in cryopreserved sperm. In fertilization trials, 0.5 g of eggs was mixed with cryopreserved sperm stored for 30 days in LN. The best motility of post-thaw sperm was achieved using 15% PG + 85% FBS (76.7 ± 8.8%); 10% PG + 90% FBS was also effective as cryodiluent. Generally, FBS gave better post-thaw motility compared to ASP. The optimum cooling rate was at 17.6 °C min⁻¹ obtained by freezing at the height of 7.5 cm surface of LN. The results obtained showed that cryopreserved sperm of brown-marbled grouper suffered slight DNA fragmentation, which resulted in significantly lower motility. However, the fertilization (90.9 ± 0.5%), hatching (64.5 ± 4.1%) and deformity rates (3.8 ± 0.2%) obtained from cryopreserved sperm showed no significant difference with fresh sperm. These findings show that the developed protocol for cryopreservation of brown-marbled grouper sperm was viable and will be useful for successful breeding and seed production of brown-marbled grouper.     

Effect of loading and vitrification solutions on survival of cryopreserved oil palm polyembryoids

The present study evaluates the effect of six loading solutions and five vitrification solutions (VS) and their time of exposure on the survival of oil palm (Elaeis guineensis) polyembryoids in liquid nitrogen (LN). In vitro grown polyembryoids of oil palm were successfully cryopreserved by vitrification with 45% survival. Individual polyembryoids, isolated from 2-month old culture, were precultured in liquid Murashige and Skoog medium supplemented with 0.5 M sucrose for 12 h and treated with a mixture of 10% (w/v) dimethyl sulphoxide (DMSO) plus 0.7 M sucrose for 30 min. Polyembryoids were then subjected to plant vitrification solution-2 (PVS2) (30% (w/v) glycerol plus 15% (w/v) EG plus 15% (w/v) DMSO plus 0.4 M sucrose) exposure for 5 min at 26 ± 2°C and subsequently plunged into LN. Thawed polyembryoids resumed growth within 8 days of culture and shoot development was recorded at 25 days of growth. Scanning electron micrograph revealed that successful regeneration of cryopreserved polyembryoids was due to stabilization of cellular integrity through optimum VS exposure.     

Phyto-constituents profiling of Luffa echinata and in vitro assessment of antioxidant,anti-diabetic,anticancer and anti-acetylcholine esterase activities

Luffa echinata Roxb. is one of the neglected medicinal plants. It is an important source of bioactive metabolites and used in several Ayurvedic formulations. In the present analysis, mature leaves and fruits were extracted with acetone, ethanol, acetonitrile, methanol and water. Phytochemicals like total phenolic (TPC), flavonoid (TFC), tannin (TTC), alkaloid (TAC) and terpenoid (TTEC) content were analysed. Further, antioxidant (AOX) activities like 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2′-azino-bis-(3-ethyl) benzothiazoline-6-sulfonic acid (ABTS) radical scavenging, metal chelating activity (MC), ferric reducing antioxidant power (FRAP) and phosphomolybdenum assay (PMA) were studied. Highest TPC and TFC (189.57 ± 1.9 mg TAE/g extract, 30.48 ± 0.7 mg CE/g extract, respectively) were reported from acetone extract of the leaves. Ethanolic fruit extract showed the highest TTC (13.79 ± 0.2 mg CE/g extract). Acetone and acetonitrile fruit extract revealed maximum TTEC (602.79 ± 3.5 mg UAE/g extract) and moderate TAC (19.96 ± 0.9 mg GE/g extract), respectively. In AOX, highest DPPH (50.52 ± 0.03 mg AAE/g extract) and ABTS (26.78 ± 0.03 mg TE/g extract) radical scavenging reported in methanolic extract of fruit; however, acetone extract of leaf showed highest FRAP (376.89 ± 1.95 mg Fe(II)/g extract) and PMA (326.54 ± 4.73 mg AAE/g extract). In contrast, aqueous extract of leaf and fruit revealed highest metal chelating activity (41.67 ± 0.49 mg EDTA/g extract). In anti-diabetic studies, acetonitrile extract of leaves and fruits exhibited appreciable inhibition of α-amylase (83.33%) and α-glycosidase (77.42%) enzymes. Similarly, acetyl cholinesterase (AChE) inhibition was highest in water (88.91%) and acetone (81.87%) extracts of leaf and fruits. Fruit extracts showed potent anticancer activity against breast (MCF-7) and colon (HT-29) cancer cell lines (LC₅₀ 329.36 and 385.17 µg/mL, respectively). RP-HPLC analysis revealed highest cucurbitacin B (CuB) (196.24 ± 1.4 mg/g DW), followed by cucurbitacin I (CuI) and cucurbitacin E (CuE) in the fruits (57.14 ± 4.9 and 2.03 ± 0.03 mg/g DW, respectively). RP-HPLC analysis of extracts revealed presence of gallic acid (GA), catechin (CA), vanillic acid (VA), chlorogenic acid (CHLA) and coumaric acid (COA), in which highest GA found in the fruits (1.26 ± 0.07 mg/g DW). Liquid chromatography and mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC–MS) analysis revealed presence of bioactive compounds from various groups. Based on the present findings, it was revealed that the fruit and leaf of L. echinata can be used as potent bioresource for natural antioxidants, anti-diabetic, and anticancer drug.     

Sperm cryostorage in a dry tank: An accurate alternative

This prospective study aimed to determine the effects of dry nitrogen cryostorage on human sperm characteristics in comparison with liquid nitrogen cryostorage. For this purpose, 42 men undergoing routine semen analysis (21 normozoospermia and 21 with altered semen parameters) were analyzed. After slow freezing, half of the straws of each sample were randomly stored in liquid and dry tanks, at the top and bottom levels of the latter. After 6 months storage, thawed samples were treated by density gradient centrifugation and sperm characteristics were compared. There was no difference in sperm progressive motility (15.1% ± 14.2% vs. 15.1% ± 12.7%; p = 0.76), sperm vitality (25.5% ± 17.7% vs. 26.2% ± 19%; p = 0.71), percentages of acrosome-reacted spermatozoa (38% ± 8.5% vs. 38.5% ± 7.4%; p = 0.53) and DNA fragmentation spermatozoa (27.3% ± 12.4% vs. 28.5% ± 12.9%, p = 0.47) after cryostorage in the dry or the liquid nitrogen tank. Moreover, we did not observe differences between either cryostorage system for normal and altered sperm samples. This lack of difference was also observed whatever the floor level of cryostorage in the dry tank. The temperature measurement of the dry tank showed a stable temperature at −194 °C throughout storage whatever the storage floor level, guaranteeing the stability of the low temperatures suitable for human sperm storage. Because of its greater safety, dry storage without contact with the liquid phase should be preferred and can be a useful alternative for the cryostorage of human sperm samples.     

Cryopreservation of giant grouper Epinephelus lanceolatus (Bloch, 1790) sperm

The grouper, Epinephelus lanceolatus, is a vulnerable species of high economic value. An effective protocol was developed for the cryopreservation of E. lanceolatus by comparing different extenders produced by mixing various cryoprotectants (dimethyl sulfoxide, methanol and glycerol) and diluents (MPRS, TS‐2, TS‐19, Cortland and Hank's). Using computer‐assisted sperm analysis (CASA) and morphological analysis, the sperm motility and fertilization rates from post‐thaw sperm were comparable to untreated controls. The results revealed that MPRS (containing 12% DMSO) or TS‐19 (containing 12% DMSO), were the optimum extenders for protecting the sperm from cryo‐damage in liquid nitrogen. The post‐thaw sperm maintained high motility (90.61 ± 3.03%) and a fertilization rate (92.27 ± 2.43%) similar (P > 0.05) to fresh sperm (94.34 ± 4% and 94.10 ± 1.87%). This study is the first to report on the successful sperm cryopreservation of E. lanceolatus and provides an important tool for repopulating this species through aquaculture.     

Antimicrobial potential of Indian Cinnamomum species

Cinnamomum is the largest genus of Lauraceae family and has been used as spices, food, and food additives by the people. Total 15 Cinnamomum species are distributed in different parts of Indian sub-continent. Different parts (leaves, stem bark, stem wood, roots, flowers, and fruits) of these species were shade-dried and used for the determination of essential oils. A total of 19 essential oils were identified and quantified from the different parts of (leaf, stem bark, stem wood, root, flower, and fruit) of 15 Cinnamomum species. The stem bark of C. altissimum was rich in the presence of essential oils (52.2 %) whereas minimum levels of essential oils were recorded in roots (17.9 %). The γ-terpinene (11.1 %) was reported as the major component essential oil in C. subavenium flowers. Methanol extract of C. camphora stem wood showed stronger lowest minimum inhibitory concentration against S. aureus (25 ± 0.01 μg/ml), H. pylori (29 ± 0.05 μg/ml), B. subtilis (31 ± 0.03 μg/ml), E. faecalis (33 ± 0.01 μg/ml), C. albicans (38 ± 0.03 μg/ml) when compared to amoxycillin (S. aureus 56 ± 0.05 μg/ml; B. subtilis 27 ± 0.04 μg/ml, E. faecalis 22 ± 0.01 μg/ml), streptomycin (H. pylori 38 ± 0.02 μg/ml) and fluconazole (C. albicans 56 ± 0.01 μg/ml). Methanolic extract of C. camphora stem wood demonstrated maximum antimicrobial activity against S. aureus, H. pylori, B. subtilis, E. faecalis and C. albicans. The essential oil of C. altissimum stem bark displayed significant lowest MIC against S. aureus (21 ± 0.03 μg/ml), E. coli (22 ± 0.03 μg/ml), E. cloacae (37 ± 0.06 μg/ml), L. monocytogenes (47 ± 0.08 μg/ml), and P. chrysogenum (101 ± 0.07 μg/ml) when compared to amoxycillin (E. coli 18 ± 0.01 μg/ml, E. cloacae 21 ± 0.05 μg/ml, L. monocytogenes 31 ± 0.03 μg/ml), and fluconazole (P. chrysogenum 101 ± 0.07 μg/ml). The essential oil of C. altissimum stem bark displayed maximum antimicrobial activity against S. aureus, E. coli, E. cloacae, L. monocytogenes, and P. chrysogenum. Cinnamomum essential oils may be used as an alternative source of antibacterial and antifungal compounds in the treatment of various types of infections.