Effects of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 on the in vitro Maturation of Porcine Oocytes

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor‐β (TGF‐β) family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expressions in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI), but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased the expression of genes involved in both oocyte maturation (c‐mos, cyclinb1 and cdc2) and cumulus expansion (has2, ptgs2, ptx3 and tnfaip6), SB431542 (a TGFβ–GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (41.3% and 74.4%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c‐mos [a regulatory subunit of mitogen‐activated protein kinase (MAPK)], and cyclinb1 and cdc2 [regulatory subunits of maturation/M‐phase‐promoting factor (MPF)]; however, SB431542 significantly decreased their mRNA levels. These data were supported by poly (A)‐test PCR and protein activity analyses. Our results show that GDF9 and BMP15 participate in cumulus expansion and that they stimulate MPF and MAPK activities in porcine oocytes during in vitro maturation.     

Bone Morphogenetic Protein‐6 (BMP‐6) Stimulates the Antrum Formation by the Regulation of its Signalling Pathway in Caprine Pre‐antral Follicles Cultured In Vitro

BMP‐6 has been found to be important to ovarian cells and oocyte, as well as to uterus. Thus, this study investigated the effect of bone morphogenetic protein (BMP‐6) and recombinant follicle‐stimulating hormone (rFSH) alone or in combination on the in vitro culture (IVC) of isolated caprine secondary follicles (Experiment 1) and the mRNA levels for BMP receptors/Smad signalling pathway (BMPR1A, BMPR2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7 and SMAD8) in vivo and in vitro using BMP‐6 (Experiment 2). Secondary follicles were cultured in αMEM⁺ alone (control medium) or supplemented with BMP‐6 at 1 or 10 ng/ml and rFSH alone or the combination of both BMP‐6 concentrations and rFSH. The results from Experiment 1 showed that the antrum formation rate was higher in the BMP‐6 at 1 ng/ml (p < 0.05) than in MEM. In Experiment 2, the mRNA expression for BMPR2, SMAD1, SMAD5 and SMAD6 was detected in non‐cultured control and after in vitro culture (MEM and 1 ng/ml BMP‐6); while the expression of SMAD7 and SMAD8 mRNA was only detected after IVC, SMAD4 was only detected in the BMP‐6 at 1 ng/ml treatment. In conclusion, the low BMP‐6 concentration positively influenced antrum formation and ensured normal mRNA expression for BMP receptor and Smads after IVC of caprine secondary follicles.     

Effects of soyabean meal‐ or whey‐based diets on lipid metabolism in weaned piglets

The present study aimed to test the hypothesis that dietary protein source influences lipid metabolism‐related parameters weaned piglets. The effects of soyabean meal (SB) and whey proteins (WP) on gene expression of several genes involved in the lipogenic process in liver, visceral (VAT) and subcutaneous (SAT) adipose tissues, plasma insulin concentration and fatty acid (FA) profile were investigated in 18 weaned piglets. Weaned piglets were fed one of two diets containing either SB or WP as the main protein source. Following a 10‐h fasting period, plasma insulin concentration and FA profile were assessed at 56 and 72 days of age, whereas gene expression in liver, VAT and SAT was assessed at 72 days of age. Plasma insulin concentration was not affected by diet, although it was 40% lower in SB fed pigs. The SB pigs had lower 14:0 (p < 0.01) and higher 18:3n‐3 (p < 0.001) levels in plasma in comparison with WP pigs. However, these changes were attributed to background differences in the dietary FA profile and not to a direct protein source effect. Gene expression of sterol regulatory element‐binding protein 1 (SREBP‐1) in liver and VAT were lower (p < 0.01 and p < 0.05, respectively) in SB compared to WP fed piglets, but no differences occurred in SAT. No changes were observed in sterol regulatory element‐binding protein 2, liver X receptor, peroxisome proliferator‐activated receptors α and γ and plasminogen activator inhibitor 1 mRNA levels, either in liver or in adipose tissues. In conclusion, dietary protein source, accompanied likely by side alterations in the dietary composition, affects lipid metabolism in pigs through the downregulation of SREBP‐1, which is a crucial determinant of lipogenic process.     

Effects of Piper sarmentosum extract on the growth performance,antioxidant capability and immune response in weaned piglets

The biological properties of Piper sarmentosum render it a potential substitute for antibiotics in livestock feed. This study evaluated the effects of P. sarmentosum extract (PSE) on the growth performance, antioxidant capability and immune response of weaned piglets. Eighty 21‐d‐old weaned piglets were selected and randomly allocated to one of four dietary treatments with five replicates of four pigs each. The dietary treatments consisted of a basal diet supplemented with 0 (T0), 50 (T50), 100 (T100) or 200 (T200) mg/kg PSE. The feeding trial lasted 4 weeks. The results revealed that the T50 group had the highest average daily gain (ADG) and average daily feed intake (ADFI) throughout the feeding trial (p < 0.05). Additionally, the T50 group had higher (p < 0.05) serum glutathione peroxidase activity (GSH‐Px) and lower (p < 0.05) serum malondialdehyde (MDA) levels than the T0 group at 4 weeks post‐weaning (p < 0.05). Serum levels of interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) decreased, while serum levels of interleukin‐4 (IL‐4), interleukin‐10 (IL‐10) and transforming growth factor‐β (TGF‐β) increased by PSE supplementation at 4 weeks post‐weaning (p < 0.05). PSE supplementation upregulated the mRNA expression of IL‐4, IL‐10 and TGF‐β and downregulated the mRNA expression of TNF‐α, IL‐1β and interleukin‐6 (IL‐6) in the ileal mucosal layer of piglets (p < 0.05). In summary, our study findings revealed that PSE supplementation improved the antioxidant capability, and reduced inflammation, which may be beneficial to weaned piglet health.     

Effects of IGF‐1 on In Vitro Culture of Bovine Preantral Follicles are Dose‐Dependent

This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF‐1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α‐MEM⁺, supplemented with different concentrations of human recombinant IGF‐1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO₂ in 24‐well plates with total replacement of the medium every 2 days. Non‐cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF‐1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF‐1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p < 0.05). Also, after 7 days of culture, IGF‐1 30 ng/ml presented a higher follicular diameter when compared to the control and other concentrations of IGF‐1 tested. Ultrastructurally, the non‐cultured control and IGF‐1 30 ng/ml, after 7 days of culture, showed conserved oocytes, nuclei and organelles. Hence, it is concluded that IGF‐1 30 ng/ml was the most efficient concentration for the development of bovine preantral follicles cultured in vitro.     

Effect of dietary Schizochytrium microalga oil on selected markers of low‐grade inflammation in rats

The objective of the present study was to evaluate a potential of Schizochytrium microalga oil to alleviate possible negative effects of high‐fat‐high‐energy diets. Forty adult male rats (Wistar Albino) were fed 7 weeks the diet containing beef tallow + evaporated sweetened milk (diet T) intended to cause mild obesity and low‐grade systemic inflammation. Consequently, the animals were divided into four groups by 10 animals each and fed either the T‐diet (control) or the diet containing 6% of safflower oil (S), 6% of fish oil (F) and 6% of Schizochytrium microalga oil (A), respectively, for another 7 weeks. The A‐diet decreased (p < 0.05) live weight to 86% and glycaemia to 85% of control, respectively; an effect of the S‐ and F‐diet on these markers was insignificant (p > 0.05). In comparison with control, higher (p < 0.05) deposition of eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) in the epididymal adipose tissue (EAT) of the A‐rats correlated with increased (p < 0.05) plasma adiponectin concentration, but it was without the effect (p > 0.05) on cellular adiponectin content in the EAT. Higher (p < 0.05) EPA+DHA deposition in the liver of the A‐rats correlated with higher expression (149% of control; p < 0.05) of the gene coding for peroxisome proliferator‐activated receptor gamma, and with lower expression (82% and 66%; p < 0.05) of the genes coding for adiponectin receptors AdipoR1 and AdipoR2; no relationship to the expression of receptor GPR120 was found. The A‐diet did not affect amount of the nuclear fraction of the nuclear factor kappa B in the liver, but increased plasma level of anti‐inflammatory cytokine TGF‐β1 (p < 0.05). The presented data agree with results of other in vivo rodent and human studies, but not with literature data regarding in vitro experiments: it can be concluded that the effects of dietary oils on inflammatory markers need further investigation.     

The localization and function of p38α mitogen‐activated protein kinase in rat oocytes

P38α mitogen‐activated protein kinase (MAPK), which is a member of the canonical MAPK family, is activated in response to various extracellular stresses and plays a role in multiple cellular processes. In this study, we investigated the expression, subcellular localization and functional roles of p38α MAPK during the meiotic maturation of rat oocytes. We found that p38α MAPK phosphorylation (p‐p38α MAPK, indicative of p38α MAPK activation) was low at the germinal vesicle (GV) stage, increased 3 hr after germinal vesicle breakdown (GVBD) and maintained its maximum at metaphase I (MI) or metaphase II (MII). The p‐p38α MAPK mainly accumulated in the GV and had no obvious expression in the nucleus. From GVBD to MII, p‐p38α MAPK was distributed in the cytoplasm around either the chromosomes or the spindle. We used SB203580, an inhibitor of p38α MAPK, to investigate the possible functional role of p38α MAPK during rat oocyte meiotic maturation. Treatment of GV stage oocytes with 20 μM SB203580 blocked p‐p38α MAPK activity, and the spindles appeared abnormal. Additionally, the rate of GVBD after 3 hr of culture with 20 μM SB203580 (58.8%) was significantly inhibited compared with the control (82.5%, p < .05), and the polar body extrusion rate after 12 hr of culture with SB203580 was also significantly decreased compared with the control (40.1% vs 73.3%, p < .05). Taken together, these data indicate that p38α MAPK may play a vital role in rat oocyte meiotic maturation.     

Polymorphisms in the promoter region of myostatin gene are associated with carcass traits in pigs

Higher average daily gain, more lean meat yield and less fat yield of porcine carcass increase selling profits for animal producers. Myostatin (MSTN), previously called GDF8, is a member of transforming growth factor‐β (TGF‐β) superfamily. It is a negative regulator for both embryonic development and adult homeostasis of skeletal muscle. In this study, the genotypes of the previously described SNPs MSTN g.435G>A and g.447A>G SNPs in 66 Duroc pigs, 33 Landrace pigs, 180 Duroc × Landrace (DL) pigs and 155 Duroc × Yorkshire × Landrace (DYL) pigs were determined by Taqman SNP Genotyping Assays. For Duroc and Landrace pigs, MSTN g.435GG/g.447AA individual had greater backfat thickness (p < 0.05) than g.435AA/g.447GG individual, whereas MSTN g.435AA/g.447GG had greater meat (p < 0.05) and meat percentage (p < 0.05) than g.435GA/g.447AG individual. For DL and DYL pigs, the MSTN g.435GG/g.447AA animals were greater in backfat at ultrasound 10th rib (p < 0.05) and carcass 10th rib (p < 0.01) than g.435AA/g.447GG individual. The MSTN g.435AA/g.447GG individual also had higher values than g.435GG/g.447AA for anterior‐end meat (p < 0.05), posterior‐end meat (p < 0.01), total meat weight (p < 0.01) and meat percentage (p < 0.01). This study confirmed evidence that MSTN g.435G>A and g.447A>G affected carcass traits in pigs. The effects of the mutated alleles were additive with the maximal effects resulting from two copies of the mutated allele. Selection for MSTN g.435A/g.447G allele is expected to increase muscle of limb and total meat production and decrease backfat thickness.     

Expression of Heparin‐Binding EGF‐Like Growth Factor (HB‐EGF) in Bovine Endometrium: Effects of HB‐EGF and Interferon‐τ on Prostaglandin Production

Heparin‐binding EGF‐like growth factor (HB‐EGF) regulates several cell functions by binding to its membrane receptor (ErbB1 and ErbB4). Experimental evidences suggest that HB‐EGF, prostaglandins (PGs) and interferon‐τ (IFN‐τ) regulate uterine function for pregnancy establishment in ruminants. In this study, the mRNA expressions of HB‐EGF, ErbB1 and ErbB4 in bovine endometrium and the effects of HB‐EGF and IFN‐τ on PGE2 and PGF2‐α production by endometrial cells were investigated. RT‐PCR analysis revealed that HB‐EGF mRNA was greater at the mid‐luteal stage than at the early and regressed luteal stages (p < 0.05). ErbB1 mRNA expression was greater at the mid‐ and late luteal stages than at the other luteal stages (p < 0.05). IFN‐τ increased the expression of HB‐EGF, ErbB1 and ErbB4 mRNA in epithelial cells (p < 0.05). HB‐EGF did not affect PGF2‐α or PGE2 production by bovine endometrial epithelial cells, but increased PGF2‐α and PGE2 production by bovine endometrial stromal cells (p < 0.05). IFN‐τ significantly decreased HB‐EGF‐stimulated PGF2‐α (p < 0.05), but not PGE2 (p > 0.05) production by stromal cells. These results indicate that HB‐EGF and its receptors expression changed in bovine endometrium throughout the oestrous cycle. IFN‐τ increased their expression in cultured endometrial cells. HB‐EGF and IFN‐τ have the ability to regulate PGs production by stromal cells and therefore may play a role in the local regulation of uterine function at the time of implantation in cattle.     

Immunohistochemical Localization of Fibroblast Growth Factor‐2 in the Sheep Ovary and its Effects on Pre‐antral Follicle Apoptosis and Development In Vitro

Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF‐2 protein expression in ovine ovaries and to verify the effect of FGF‐2 on the morphology, apoptosis and growth of ovine pre‐antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α‐MEM⁺) alone or supplemented with FGF‐2 at different concentrations (1, 10, 50, 100 or 200 ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF‐2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10 ng/ml FGF‐2, and significantly higher than those observed in 50, 100 or 200 ng/ml FGF‐2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200 ng/ml FGF‐2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200 ng/ml FGF‐2 (p < 0.05). However, the percentage of apoptotic cells only decreased (p < 0.05) in ovarian tissues cultured in 1 or 10 ng/ml FGF‐2 compared with the control medium and other FGF‐2 treatments. In conclusion, this study demonstrated the presence of FGF‐2 in ovine ovaries. Furthermore, 10 ng/ml FGF‐2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis.     

Methyl donors dietary supplementation to gestating sows diet improves the growth rate of offspring and is associating with changes in expression and DNA methylation of insulin‐like growth factor‐1 gene

The study aimed to investigate the effects of maternal dietary methyl donors on the performance of sows and their offspring, and the associated hepatic insulin‐like growth factor‐1 (IGF‐1) expression of the offspring. A total of 24 multiparous sows were randomly fed the control (CON) or the CON diet supplemented with methyl donors (MD) at 3 g/kg betaine, 15 mg/kg folic acid, 400 mg/kg choline and 150 μg/kg VB₁₂, from mating until delivery. After farrowing, sows were fed a common lactation diet through a 28‐days lactation period and six litters per treatment were selected to be fed until at approximately 110 kg BW. Maternal MD supplementation resulted in greater birthweight (p < 0.05) and increased the piglet weights (p < 0.01) and litter weights (p < 0.05) at the age of day 28, compared with that in CON group. The offspring pigs in the MD group had greater ADG (p < 0.05) and tended to lower F:G ratio (p = 0.07) compared with that of CON group from day 28 to 180 of age. The offspring pigs from MD group had greater serum IGF‐1 concentrations and expressions of hepatic IGF‐1 gene and muscular IGF‐1 receptor (IGF‐1r) protein at birth (p < 0.05), and greater hepatic IGF‐1 protein (p = 0.03) and muscular IGF‐1r gene expressions (p < 0.05) at slaughter, than that from the CON group. Moreover, the methylation at the promoter of IGF‐1 gene in the liver of newborn piglets and finishing pigs was greater in the MD group than that of the CON group (p < 0.05). In conclusion, maternal MD supplementation throughout gestation could enhance the birthweight and postnatal growth rate of offspring, associated with an increased expression of the IGF‐1 gene and IGF‐1r, as well as the altered DNA methylation of IGF‐1 gene promotor.     

Combining ultrasound and lactobacilli treatment for high‐fat‐diet‐induced obesity in mice

Chronic systemic lipopolysaccharide‐induced inflammation can cause obesity. In animal experiments, lactobacilli have been shown to inhibit obesity by modifying the gut microbiota, controlling inflammation and influencing the associated gene expression. A previous study found that high‐fat‐diet‐induced (HFD) obesity was suppressed by lactobacilli ingestion in rats via the inhibition of parasympathetic nerve activity. This study explored the combined use of lactobacilli ingestion and ultrasound (US) to control body weight and body fat deposition in HFD mice over an 8‐week experimental period. Male C57BL/6J mice received an HFD during treatment and were randomly divided into four groups: (i) control group (H), (ii) lactobacilli alone (HB), (iii) US alone (HU) and (iv) lactobacilli combined with US (HUB). The US was targeted at the inguinal portion of the epididymal fat pad on the right side. At the 8th week, body weight had decreased significantly in the HUB group (15.56 ± 1.18%, mean ± SD) group compared with the HU (26.63 ± 0.96%) and H (32.62 ± 5.03%) groups (p < 0.05). High‐resolution microcomputed tomography (micro‐CT) scans revealed that the reduction in total body fat volume was significantly greater in the HUB group (69%) than in the other two experimental groups (HB, 52%; HU, 37%; p < 0.05). The reductions in the thickness of the subcutaneous epididymal fat pads were significantly greater in the HUB group (final thickness: 340 ± 7 μm) than in the H (final thickness: 1150 ± 21 μm), HB (final thickness: 1060 ± 18 μm) and HU (final thickness: 370 ± 5 μm) groups (all p < 0.05). Combination therapy with lactobacilli and US appears to enhance the reduction in body weight, total and local body fat deposition, adipocyte size and plasma lipid levels over an 8‐week period over that achieved with lactobacilli or US alone in HFD mice. These results indicate that US treatment alone can reduce hyperlipidemia in HFD mice.     

Effect of dietary threonine on laying performance and intestinal immunity of laying hens fed low‐crude‐protein diets during the peak production period

Threonine (Thr) may be a limiting amino acid for laying hens fed diets with lowered protein level. An experiment was conducted to examine laying performance, and the intestinal immune function of laying hens provided diets varying in digestible Thr levels. Lohmann Brown laying hens (n = 480), 28 weeks of age, were allocated to six dietary treatments, each of which included five replicates of 16 hens. Dietary crude protein (CP) 16.18% diet was offered as the positive control diet. L‐Thr was added to the negative diet (14.16% CP) by 0, 1.0, 2.0, 3.0 and 4.0 g/kg, corresponding 0.44%, 0.43%, 0.49%, 0.57%, 0.66% and 0.74% digestible Thr. At 40 weeks, a reduction in CP level decreased laying performance (p < 0.05). In the low CP, increasing dietary Thr increased (p < 0.05) egg production and egg mass and rose to a plateau between 0.57% and 0.66%. The hens fed 0.66% Thr showed the lowest value (p < 0.05) of feed conversion ratio (FCR). Serum level of uric acid showed the lowest values (p < 0.05) at 0.57–0.66%. In addition, serum‐free Thr maximized (p < 0.05) between 0.66% and 0.74%. Digestive trypsin activity decreased (p < 0.05) when hens fed the low‐CP diet compared with hens fed CP (16.18%) and hens fed 0.57–0.66%. Expressions of ileal MUC2 mRNA maximized (p < 0.05) at 0.66% Thr. Occludin mRNA increased with increasing Thr level (p < 0.05). sIgA mRNA reached to the maximum level (p < 0.05) at 0.66% and 0.74% Thr. INF‐γ mRNA reached to the lowest level (p < 0.05) at 0.65%. Expressions of ileal IL‐2, IL‐6, IL‐1β mRNA decreased with increasing Thr level (p < 0.05). In conclusion, Thr supplementation resulting in optimal laying performance and stimulated the mucosal immune system, suggesting that it is a limiting amino acid in the low‐crude‐protein diet of laying hens during the peak production period.     

Effects of Dietary Supplementation of β‐hydroxy‐β‐methylbutyrate on Sow Performance and mRNA Expression of Myogenic Markers in Skeletal Muscle of Neonatal Piglets

The effects of dietary β‐hydroxy‐β‐methylbutyrate (HMB) supplementation during gestation on reproductive performance of sows and the mRNA expression of myogenic markers in skeletal muscle of neonatal pigs were determined. At day 35 of gestation, a total of 20 sows (Landrace × Yorkshire, at third parity) were randomly assigned to two groups, with each group receiving either a basal diet or the same diet supplemented with 4 g/day β‐hydroxy‐β‐methylbutyrate calcium (HMB‐Ca) until parturition. At parturition, the total and live litter size were not markedly different between treatments, however, the sows fed HMB diet had a decreased rate of stillborn piglets compared with the sows fed the control (CON) diets (p < 0.05). In addition, piglets from the sows fed HMB diet tended to have an increased birth weight (p = 0.08), and a reduced rate of low birth weight piglets (p = 0.05) compared with piglets from the CON sows. Nevertheless, lower feed intake during lactation was observed in the sows fed the HMB diet compared with those on the CON diet (p < 0.01). The relative weights of the longissimus dorsi (LD) and semitendinosus (ST) muscle were higher (p < 0.05) in neonatal pigs from the HMB than the CON sows. Furthermore, maternal HMB treatment increased the mRNA levels of the myogenic genes, including muscle regulatory factor‐4 (MRF4, p < 0.05), myogenic differentiation factor (MyoD) and insulin‐like growth factor‐1 (IGF‐1, p < 0.01). In conclusion, dietary HMB supplementation to sows at 4 g/day from day 35 of gestation to term significantly improves pregnancy outcomes and increases the expression of myogenic genes in skeletal muscle of neonatal piglets, but reduces feed intake of sows during lactation.     

Lack of glucuronidation products of trans‐resveratrol in plasma and urine of cats

Resveratrol has generated interest in cats due to reported health benefits. Cats have low activity of β‐glucuronidase, and we hypothesized they could not form two common resveratrol metabolites, resveratrol‐3‐O‐glucuronide and resveratrol‐4′‐O‐glucuronide. Resveratrol, 3 mg/cat/day, was given orally to intact male (n = 5) and female cats (n = 5) for 4 weeks. A control group (8 intact males) was used for comparison. Plasma and urine were collected weekly and analysed using high‐pressure liquid chromatography coupled with tandem mass spectrometry. Resveratrol and resveratrol‐3‐O‐sulphate, but no glucuronide metabolites, were detected in plasma and urine. Median (range 10–90th percentile) plasma resveratrol for control and treatment groups was 0.46 ng/ml (0.02–1.74 ng/ml) and 0.96 ng/ml (0.65–3.21 ng/ml). Median (range) plasma resveratrol‐3‐O‐sulphate for control and treatment groups was 6.32 ng/ml (2.55–10.29 ng/ml) and 11.45 ng/ml (1.47–53.29 ng/ml). Plasma resveratrol differed from control in week 4, while plasma resveratrol‐3‐O‐sulphate was different in all weeks (p < 0.05). Median (range) urine resveratrol for control and treatment groups was 0.28 ng/ml (0.05–1.59 ng/ml) and 19.98 ng/ml (8.44–87.54 ng/ml). Median (range) urine resveratrol‐3‐O‐sulphate for control and treatment groups was 26.71 ng/ml (10.50–75.58 ng/ml) and 108.69 ng/ml (11.83–231.05 ng/ml). All time points for urine resveratrol and resveratrol‐3‐O‐sulphate were significantly different from control (p < 0.05), except for weeks 1, 3 and 4 for resveratrol. The results support our hypothesis that cats are unlikely able to glucuronidate resveratrol, most likely due to a reduction in the activity of β‐glucuronidase.     

Improved milk production through PG–PL system by provision of in‐house shelter management in lactating Murrah buffaloes during winter season

Comprehensive information on the role of β‐casein and plasminogen–plasmin (PG–PL) system in milk secretion of Murrah buffaloes during winter season is lacking, although effects of cold stress can be ameliorated to an extent by altering microclimate at farm level. Hence, this study was aimed to determine the changes in productivity along with PG–PL system of milk, plasma hormones and metabolites of buffaloes during winter (December–January) season under two different management systems. Average minimum temperature and wind chill index during this season were 7.02 and 12.74 °C respectively. Buffaloes were divided in two groups of six animals each: control and treatment, where treatment group animals were placed in‐house with floor bedding of paddy straw and the control group animals in loose housing system without straw bedding. Physiological responses were recorded, and milk and blood samples were collected at weekly intervals for six‐week experimental period. Under in‐house management system, buffaloes experienced better comfort by alleviating environmental stress as their physiological responses such as respiration rate and pulse rate were significantly reduced (p < 0.01) as compared to the control, which subsequently resulted higher milk yield by 9.92% (p < 0.05). Analysis of milk samples revealed higher concentration of plasminogen (10.6 vs. 8.05 μg/ml; p < 0.01) and β‐casein (p < 0.05), and lower plasmin level (0.299 vs. 0.321 μg/ml; p < 0.05) in buffaloes under treatment group. It was also found that plasma cortisol, glucose and non‐esterified fatty acids levels were higher (p < 0.01) in control group as compared to the treatment animals by 13.6%, 8.14% and 12.6% respectively. However, milk composition, growth hormone, epinephrine and norepinephrine level in plasma were similar in both the groups. Hence, it may be concluded that provision of in‐house shelter management with floor bedding of paddy straw during winter was effective to minimize environmental stress and improved milk production through manipulation of PG–PL system in buffaloes.     

Alpha‐Linolenic Acid Supplementation in Tris Extender Can Improve Frozen–Thawed Bull Semen Quality

The study was conducted to evaluate the effects of α ‐linolenic acid (ALA) on frozen–thawed quality and fatty acid composition of bull sperm. For that, twenty‐four ejaculates obtained from three bulls were diluted in a Tris extender containing 0 (control), 3, 5, 10 and 15 ng/ml of ALA. Extended semen was incubated at 37°C for 15 min, to allow absorption of ALA by sperm cell membrane. The sample was chilled for 2 h, packed into 0.25‐ml straws and frozen in liquid nitrogen for 24 h. Subsequently, straws were thawed and evaluated for total sperm motility (computer‐assisted semen analysis), membrane functional integrity (hypo‐osmotic swelling test), viability (eosin‐nigrosin), fatty acid composition (gas chromatography) and lipid peroxidation (thiobarbituric acid‐reactive substances (TBARS)). A higher (p < 0.05) percentage of total sperm motility was observed in ALA groups 5 ng/ml (47.74 ± 07) and 10 ng/ml (44.90 ± 0.7) in comparison with control (34.53 ± 3.0), 3 ng/ml (34.40 ± 2.6) and 15 ng/ml (34.60 ± 2.9). Still, the 5 ng/ml ALA group presented a higher (p < 0.05) percentage of viable sperms (74.13 ± 0.8) and sperms with intact membrane (74.46 ± 09) than all other experimental groups. ALA concentration and lipid peroxidation in post‐thawed sperm was higher in all treated groups when compared to the control group. As such, the addition of 5 ng/ml of ALA to Tris extender improved quality of frozen–thawed bull spermatozoa.     

Effect of Physiologically Relevant Heat Shock on Development,Apoptosis and Expression of Some Genes in Buffalo (Bubalus bubalis) Embryos Produced In Vitro

For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8‐cell, 16‐cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8–16‐cell and blastocyst stages, relative mRNA abundance of stress‐related genes HSP 70.1 and HSP 70.2 and pro‐apoptotic genes CASPASE‐3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress‐related gene HSF1. Expression level of anti‐apoptotic genes BCL‐XL and MCL‐1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8–16‐cell and blastocyst stages. Among the genes related to embryonic development, at 8–16‐cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR‐1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress‐, apoptosis‐ and development‐related genes.