Fatty acid concentration of plasma, muscle, adipose and liver from beef heifers fed an encapsulated n-3 polyunsaturated fatty acid supplement.

Increasing the content of polyunsaturated fat in the human diet is a priority for reducing cardiovascular disease and cancer risks. Beef has the potential to contribute to the polyunsaturated fat content in the human diet; however, ruminants cannot synthesise many long-chain fatty acids de novo; they require dietary supplementation. The objectives of the current study were to evaluate (i) the effect of a partially rumen protected n-3 long-chain polyunsaturated fatty acid (LC-PUFA) dietary supplement on the fatty acid composition of muscle (Longissimus dorsi), adipose and liver tissues of beef heifers and (ii) the usefulness of blood plasma as a predictor of tissue concentrations of specific fatty acids. Charolais crossbred heifers (n = 20) were assigned to one of two isolipid dietary treatments namely palmitic acid (control) or an n-3 LC-PUFA supplement for a 91-day period. Blood plasma and adipose tissue samples were taken to determine the temporal effect of these diets on fatty acid composition (days 0, 10, 35 and 91), while liver and muscle samples were taken following slaughter. Dietary lipid source did not influence animal growth rate or body condition score. At day 91, the percentage differences between control and n-3 LC-PUFA heifers in concentrations of eicosapentaenoic acid were +61, +176 and +133 % in liver, muscle and adipose, respectively. For docosahexaenoic acid, at the same time point, the percentage differences were +57, +73 and +138 % for liver, muscle and adipose, respectively. Medium-to-strong positive correlation coefficients were evident for liver and plasma fatty acids, in particular, there were positive relationships with concentrations of total saturated fatty acid (SFA), total n-6 PUFA and total n-3 PUFA. This trend also extended to both the ratio of PUFA to SFA (slope (ß1) = 0.56 ±â€¯0.167, intercept (ß0) = 0.56, R2 = 0.61, P < 0.05) and the ratio of n-6 to n-3 PUFA (ß1 = 0.15 ±â€¯0.054, ß0 = 0.24, R2 = 0.52, P < 0.05). A strong correlation was also detected in the ratio of n-6 to n-3 in plasma and muscle tissue of heifers fed the n-3 LC-PUFA diet (ß1 = 0.53 ±â€¯0.089, ß0 = -0.31, R2 = 0.83, P < 0.001). The results of this study show that the n-3 LC-PUFA can be readily increased through targeted supplementation and that plasma concentrations of n-3 LC-PUFA are useful predictors of their concentrations in a number of economically important tissues.

Effect of dietary n-3 polyunsaturated fatty acid supplementation and post-insemination plane of nutrition on systemic concentrations of metabolic analytes, progesterone, hepatic gene expression and embryo development and survival in beef heifers.

Nutrition, and particularly dietary energy intake, plays a fundamental role in reproductive function in cattle. There is some evidence that supplemental omega-3 dietary polyunsaturated fatty acids (n-3 PUFA) can exert positive effects on fertility. The objectives of this study were to evaluate the effect of dietary n-3 PUFA supplementation, post-insemination energy plane of nutrition and their interaction on embryo survival in cattle. Crossbred beef heifers (n = 185) were individually offered barley straw ad libitum and 6 kg DM of concentrate supplemented with either a rumen-protected source of saturated fatty acid (palmitic; control, CON) or a partially rumen-protected n-3 PUFA-enriched supplement (n-3 PUFA). Estrous was synchronised using two injections of PG administered at 11-d intervals and following artificial insemination (AI = Day 0) 179 heifers exhibiting oestrus were inseminated and assigned to one of two dietary treatments: (i) remain on their pre-insemination high dietary plane of nutrition (High) or (ii) restricted to 0.6 × estimated maintenance energy requirements (Low) in a 2 × 2 factorial design. The heifers were then maintained on their assigned diets until slaughter and embryo recovery on Day 16 (n = 92) or pregnancy diagnosis by ultrasound scanning at Day 30 post-AI (n = 87). Plasma concentrations of fatty acids, metabolites, insulin, progesterone (P4) and insulin-like growth factor 1 (IGF-1) were measured at appropriate intervals. Hepatic expression of mRNA for aldo-keto reductase (AKR1C), cytochrome P450 2C (CYP 2C) and cytochrome P450 3A (CYP 3A) was examined. The n-3 PUFA supplementation increased plasma n-3 PUFA concentration (P < 0.05) and reduced n-6: n-3 PUFA ratio (P < 0.05). Plasma IGF-1 was higher for n-3 PUFA relative to the CON (P < 0.05) and for High compared with Low plane of nutrition post-AI (P < 0.05) groups. A low plane of nutrition post-AI increased plasma concentrations of progesterone from Days 7-16 after insemination (P < 0.001) but reduced embryo length (P < 0.001). Supplementation with n-3 PUFA reduced and tended to reduce hepatic expression of CYP2C (P = 0.01) and CYP3A (P = 0.08), respectively. However, while dietary n-3 PUFA supplementation and an abrupt reduction in nutrient status following insemination elevated plasma concentrations of n-3 PUFA and mid and late phase P4, respectively, there was no effect of either PUFA supplementation or post-insemination plane of nutrition on embryo survival.

Dietary polyunsaturated fatty acid supplementation of young post-pubertal dairy bulls alters the fatty acid composition of seminal plasma and spermatozoa but has no effect on semen volume or sperm quality.

The aim of this study was to examine the effects of dietary supplementation with rumen protected n-6 or n-3 polyunsaturated fatty acids (PUFA) on the quantity and quality of semen from young post-pubertal dairy bulls. Pubertal Holstein-Friesian (n = 43) and Jersey (n = 7) bulls with a mean ± s.e.m. age and bodyweight of 420.1 ± 5.86 days and 382 ± 8.94 kg, respectively, were blocked on breed, weight, age and semen quality (based on the outcomes of two pre-trial ejaculates) and randomly assigned to one of three treatments: (i) a non-supplemented control (CTL, n = 15), (ii) rumen-protected safflower (SO, n = 15), (iii) rumen-protected n-3 PUFA-enriched fish oil (FO, n = 20). Bulls were fed their respective diets, ad libitum for 12 weeks; individual intakes were recorded using an electronic feeding system for the initial 6 weeks of the feeding period. Semen was collected via electro-ejaculation at weeks -2, -1, 0, 7, 10, 11 and 12 relative to the beginning of the trial period (week 0). On collection, semen volume, sperm concentration and progressive linear motility (PLM) were assessed. On weeks -2, -1, 0, 10, 11, 12, semen was packaged into 0.25 mL straws and frozen using a programmable freezer. On weeks -1, 7 and 11; a sub-sample of semen was separated into sperm and seminal plasma, by centrifugation and stored at - 20 °C until analysis of lipid composition. Semen from 10 bulls per treatment were used for post-thaw analysis at weeks 10, 11 and 12 (3 straws per ejaculate). Sperm motility was analysed by computer assisted semen analysis (CASA). In addition, membrane fluidity, acrosome reaction and oxidative stress were assessed using flow cytometry. Sperm from bulls fed SO had a 1.2 fold higher total n-6 PUFA content at week 11 compared to week -1 (P < 0.01) while bulls fed FO had a 1.3 fold higher total n-3 PUFA content, in sperm by week 11 (P < 0.01). There was no effect of diet on semen volume, concentration or PLM of sperm when assessed either immediately following collection or post-thawing. Membrane fluidity and oxidative stress of sperm were also not affected by diet. The percentage of sperm with intact-acrosomes was lower in CTL bulls compared to those fed SO (P < 0.01). In conclusion, while the lipid composition of semen was altered following dietary supplementation with either n-6 or n-3 based PUFA, this did not lead to measurable improvements in the quantity or quality of semen produced by young post-pubertal dairy bulls.

The effect of dietary n-3 polyunsaturated fatty acids supplementation of rams on semen quality and subsequent quality of liquid stored semen.

The objective of this study was to examine the effect of dietary n-3 polyunsaturated fatty acid (PUFA) supplementation of rams on semen quality and subsequent sperm function of liquid stored semen. Mature rams of proven fertility were individually housed and were blocked according to breed, body weight, and body condition score and randomly allocated within block to one of two dietary treatments (N = 7 per treatment). Rams were offered a base diet of hay and concentrate, with the concentrate enriched with either: (1) saturated palmitic acid (CON) or (2) high n-3 PUFA fish oil (FO) supplements. Both lipid supplements were added at 2% (wt/wt) of the total diet as fed and both were partially rumen-protected. The animals were fed their respective diets for a total of 9 weeks and blood samples were collected on weeks 0 (pre-experimental), 4, and 9, relative to initial allocation of diet (week 0), for measurement of plasma concentration of fatty acids, metabolites, insulin like growth factor 1 (IGF-1) and insulin. Semen was collected from each ram (on 1 day in each week) in weeks 4, 5, 7, 8, and 9, and each ejaculate was assessed for volume, wave motion, and concentration of sperm, after which it was diluted in a skim milk-based extender and stored at 4 °C. A second ejaculate was collected on weeks 4, 7, and 9, centrifuged, and the sperm frozen for subsequent lipid analysis. A sample of semen from each ram was assessed at 24, 48, and 72 hours after collection for sperm progressive linear motion, ability to penetrate artificial mucus, and the ability to resist lipid peroxidation (at 24 and 48 hours only) using the thiobarbituric acid reactive substances assay. There was no effect of diet on plasma insulin concentrations or on any of the metabolites measured, however, there was a diet by week interaction for plasma IGF-1 concentration (P < 0.05). This was manifested as the FO supplemented rams having higher IGF-1 concentrations on week 9 compared with the control treatment (P < 0.05), but not at the earlier sampling dates. Compared with the pre-experimental values, supplementation with FO increased plasma concentrations of total n-3 PUFAs by 3.1-fold and decreased n-6 PUFA concentrations by 1.84-fold. Consequently, the ratio of n-6 to n-3 PUFA was decreased in the FO-supplemented rams (P < 0.001). Dietary supplementation with FO increased the concentration of eicosapentaenoic acid in sperm from week 4 to 9 by 2.7-fold (P < 0.05) leading to a 1.5-fold increase in total n-3 PUFA in the same period. Ejaculates collected from rams supplemented with FO yielded a higher semen concentration (P < 0.05), however, there was no difference between diets on any of the other semen quality parameters including semen volume, wave motion, progressive linear motion, ability to penetrate artificial mucus, or ability to resist lipid peroxidation. In conclusion, dietary supplementation of rams with n-3 PUFA successfully increased the n-3 PUFA content of plasma and sperm but has limited effects on the quality of liquid stored semen.

Responses of anaerobic rumen fungal diversity (phylum Neocallimastigomycota) to changes in bovine diet.

AIMS: Anaerobic rumen fungi (Neocallimastigales) play important roles in the breakdown of complex, cellulose-rich material. Subsequent decomposition products are utilized by other microbes, including methanogens. The aim of this study was to determine the effects of dietary changes on anaerobic rumen fungi diversity. METHODS AND RESULTS: Altered diets through increasing concentrate/forage (50 : 50 vs 90 : 10) ratios and/or the addition of 6% soya oil were offered to steers and the Neocallimastigales community was assessed by PCR-based fingerprinting with specific primers within the barcode region. Both a decrease in fibre content and the addition of 6% soya oil affected Neocallimastigales diversity within solid and liquid rumen phases. The addition of 6% soya oil decreased species richness. Assemblages were strongly affected by the addition of 6% soya oil, whereas unexpectedly, the fibre decrease had less effect. Differences in volatile fatty acid contents (acetate, propionate and butyrate) were significantly associated with changes in Neocallimastigales assemblages between the treatments. CONCLUSIONS: Diet clearly influences Neocallimastigales assemblages. The data are interpreted in terms of interactions with other microbial groups involved in fermentation processes within the rumen. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge on the influence of diet on anaerobic fungi is necessary to understand changes in microbial processes occurring within the rumen as this may impact on other rumen processes such as methane production.

The effect of dietary concentrate and soya oil inclusion on microbial diversity in the rumen of cattle.

AIMS: Methane emissions from ruminants are a significant contributor to global greenhouse gas production. The aim of this study was to examine the effect of diet on microbial communities in the rumen of steers. METHODS AND RESULTS: The effects of dietary alteration (50 : 50 vs 90 : 10 concentrate-forage ratio, and inclusion of soya oil) on methanogenic and bacterial communities in the rumen of steers were examined using molecular fingerprinting techniques (T-RFLP and automated ribosomal intergenic spacer analysis) and real-time PCR. Bacterial diversity was greatly affected by diet, whereas methanogen diversity was not. However, methanogen abundance was significantly reduced (P = 0.009) in high concentrate-forage diets and in the presence of soya oil (6%). In a parallel study, reduced methane emissions were observed with these diets. CONCLUSIONS: The greater effect of dietary alteration on bacterial community in the rumen compared with the methanogen community may reflect the impact of substrate availability on the rumen bacterial community. This resulted in altered rumen volatile fatty acid profiles and had a downstream effect on methanogen abundance, but not diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding how rumen microbial communities contribute to methane production and how these microbes are influenced by diet is essential for the rational design of methane mitigation strategies from livestock.

Methane emissions, feed intake, and performance of finishing beef cattle offered maize silages harvested at 4 different stages of maturity.

This experiment aimed to quantify the methane emissions and intake, digestibility, performance, and carcass characteristics of finishing beef cattle offered maize (Zea mays) silages harvested at 1 of 4 sequential stages of maturity and to relate these values to those obtained from animals offered an ad libitum concentrate-based diet. Sixty continental crossbred steers with a mean initial BW of 531 kg (SD 23.8) were blocked (n = 12 blocks) according to BW and allocated from within block to 1 of 5 dietary treatments in a randomized complete block design: maize silage harvested on September 13 (DM = 277 g/kg), maize silage harvested on September 28 (DM = 315 g/kg), maize silage harvested on October 9 (DM = 339 g/kg), maize silage harvested on October 23 (DM = 333 g/kg), and ad libitum concentrates (ALC). Diets based on maize silage were supplemented with 2.57 kg of concentrate DM daily, and ALC diets were supplemented with 1.27 kg of grass silage DM daily. Silage and total DMI were greater (P = 0.004) with maize silage harvested on September 28 than with any other treatment, which in turn did not differ. Advancing maize maturity at harvest did not affect BW or carcass gain, with the ALC diet exhibiting greater (P = 0.036) rates of carcass gain than any of the maize silage-based treatments. Apparent in vivo digestibility, determined using the AIA indigestible marker technique, was not affected by harvest maturity, with no linear or quadratic trends being identified. Digestibility of DM from the ALC diet was greater (P < 0.001) than with any of the maize silage treatments. Starch digestibility did not differ across maize silage maturities; however, a linear (P = 0.009) decrease in NDF digestibility was observed. Methane emissions, (g/d) measured using the sulfur hexafluoride tracer technique, were not affected by maize silage maturity. Methane emissions relative to DMI tended (P = 0.05) to decline with advancing maize silage maturity, with a similar decline observed when methane was expressed per kilogram of carcass gain. Advancing maize maturity did not result in significant linear or quadratic responses in methane output proportional to GE intake. The ALC diet resulted in less methane output than the maize silage treatments irrespective of the unit of expression. In conclusion, advancing maize harvest maturity did not affect beef cattle performance but reduced methane output relative to DMI and carcass gain. Cattle offered ALC exhibited greater rates of BW gain and less emission of methane compared with cattle offered any of the maize silage treatments.

Effect of DL-malic acid supplementation on feed intake, methane emissions, and performance of lactating dairy cows at pasture.

The objective of this study was to determine the effect of dietary dl-malic acid (MA) supplementation on feed intake, methane (CH(4)) emissions, and performance of mid lactation Holstein-Friesian cows at pasture. Twenty-four (6 primiparous and 18 multiparous) mid- to late-lactation cows (206 +/- 65 d in milk) grazing a mixed-species grass sward were blocked on parity, days in milk, and pretrial milk yield, and randomly allocated within block to 1 of 2 dietary treatments offered twice daily at milking in 2 equal portions (6 kg/d in total): a control concentrate (0 g/d of MA) and a concentrate supplemented with MA (480 g/d of MA) over a 6-wk period. Cows were allowed a 3-wk acclimation period followed by a 5-d CH(4) measurement period. Enteric CH(4) emissions were estimated using the sulfur hexafluoride tracer gas technique, and herbage intake was measured using the n-alkane technique. Dietary supplementation with MA did not affect voluntary intake of herbage or total dry matter intake, body weight gain, milk yield, fat-corrected milk yield, or daily CH(4) production. These results suggest that there is little benefit to be gained from the dietary supplementation of dairy cows at pasture with MA at least within the inclusion rates used in this study.

Effect of level and duration of dietary n-3 polyunsaturated fatty acid supplementation on the transcriptional regulation of Delta9-desaturase in muscle of beef cattle.

The objective of this study was to examine the effect of level and duration of feeding of an n-3 PUFA-enriched fish oil (FO) supplement in combination with soybean oil (SO) on the transcriptional regulation of Delta(9)-desaturase gene expression in bovine muscle. Beef bulls (n = 40) were assigned to 1 of 4 iso-lipid and isonitrogenous concentrate diets fed for ad libitum intake for a 100-d finishing period. Concentrates were supplemented with one of the following: 1) 6% SO (CON); 2) 6% SO + 1% FO (FO1); 3) 6% SO + 2% FO (FO2); or 4) 8% palmitic acid for the first 50 d and 6% SO + 2% FO for the second 50 d [FO2(50)]. Samples of LM were harvested and concentrations of fatty acids were measured. Total RNA was isolated and the gene expression of Delta(9)-desaturase was determined. The mRNA expression of putative regulators of Delta(9)-desaturase gene expression, sterol regulatory element binding protein-1c (SREBP-1c) and peroxisome proliferator activated receptor-alpha (PPAR-alpha), were also measured in the CON and FO2 groups. Expression of mRNA for Delta(9)-desaturase was decreased (P < 0.05) 2.6-, 4.4-, and 4.9-fold in FO1, FO2(50), and FO2 compared with CON, respectively. Expression of Delta(9)-desaturase mRNA tended to be reduced (P = 0.09) by increasing FO from 1 to 2%, but was not affected by duration of supplementation (P > 0.24). Expression of mRNA for SREBP-1c was decreased 2-fold in FO2 compared with CON (P < 0.05), whereas expression of PPAR-alpha was not affected (P > 0.30). There was a positive relationship between Delta(9)-desaturase and SREBP-1c gene expression (P < 0.01), but the expression of both genes was negatively related to tissue concentrations of n-3 PUFA (P < 0.05) and positively related to concentration of n-6 PUFA (P < 0.01). Simultaneous enhancement of tissue concentrations of CLA and n-3 PUFA concentrations in bovine muscle may be hindered by negative interactions between n-3 PUFA and Delta(9)-desaturase gene expression, possibly mediated through reduced expression of SREBP-1c.

Effect of level of eicosapentaenoic acid on the transcriptional regulation of Δ-9 desaturase using a novel in vitro bovine intramuscular adipocyte cell culture model.

Ruminant fat is often perceived as having a negative impact on human health; however, the composition of the fat is under complex biochemical control and can be improved through strategic manipulation of the animal's diet. There were two major objectives of this study, namely (i) to develop and validate a primary bovine intramuscular adipocyte cell line and (ii) to examine the effect of eicosapentaenoic acid (EPA) on the transcriptional regulation of Δ-9 desaturase in vitro using the novel cell line. Intramuscular adipose tissue was obtained from the Musculus longissimus thoracis of a beef heifer. Mature adipocytes were isolated and cultured, and subsequently harvested and evaluated for lipid accumulation and the expression of genes regulating key functional adipocyte protein markers at passages 10, 20 and 30. Isolated cells were shown to accumulate lipid in culture over time. Fatty acid analysis by gas chromatography was carried out at passage 30. Thirteen fatty acids ranging from tetradecanoic acid (C14:0) to the polyunsaturated fatty acid, docosahexaenoic acid (C22:6), were easily detected and measured. High-quality total RNA was isolated from adipocytes and the expression of peroxisome proliferator-activated receptor-γ, fatty acid synthase, fatty acid-binding protein-4, adipocyte lipid-binding protein, CD36, Δ-9 desaturase, sterol regulatory element-binding protein (SREBP), microsomal triglyceride transfer protein and leptin genes were identified by reverse transcriptase-PCR and sequence analysis. Expression of the negative control, liver-specific hepatocyte nuclear factor-1alpha, was not detected. Adipocytes were subsequently incubated in medium containing 0, 50 or 100 µM EPA for 24 h. Increasing the EPA concentration of the culture media led to a linear increase in adipocyte EPA concentration (P < 0.01). Expression of Δ-9 desaturase mRNA was decreased five- and seven-fold, respectively, following 50 and 100 µM EPA incubation compared to the control. Gene expression of SREBP-1c was decreased by 6- and 18-fold in cells supplemented with 50 and 100 µM EPA, respectively, compared to the control. Regression analysis showed a negative linear relationship between EPA concentration and the gene expression of both Δ-9 desaturase (P < 0.001) and SREBP-1c (P < 0.001), while a significant positive relationship was observed between Δ-9 desaturase and SREBP-1c gene expression (P < 0.001). This is the first report demonstrating that EPA treatment of bovine intramuscular adipocyte cells decreased gene expression of both Δ-9 desaturase and SREBP-1c in vitro. The bovine adipocyte cell line developed here is an important resource for future studies facilitating less-expensive, rapid screening of research hypotheses and circumventing the limitations associated with the use of experimental animals including cost, inter-animal variation, pre-experimental management and ethics.

Effect of DL-malic acid supplementation on feed intake, methane emission, and rumen fermentation in beef cattle.

The objective of this study was to determine the effect of dietary concentration of dl-malic acid (MA) on DMI, CH(4) emission, and rumen fermentation in beef cattle. Two Latin square experiments were conducted. In Exp. 1, six beef heifers (19 +/- 1 mo old) were assigned in a duplicated Latin square to 1 of 3 dietary concentrations of MA on a DMI basis (0%, MA-0; 3.75%, MA-3.75; or 7.5%, MA-7.5) over 3 periods. In Exp. 2, four rumen-fistulated steers (48 +/- 1 mo old) were assigned to 1 of 4 dietary concentrations of MA (0%, MA-0; 2.5%, MA-2.5; 5.0%, MA-5.0; or 7.5%, MA-7.5) on a DMI basis, over 4 periods. Both experimental diets consisted of grass silage and pelleted concentrate (containing MA). Silage was fed ad libitum once daily (a.m.), whereas concentrate was fed twice daily (a.m. and p.m.) with the aim of achieving a total DMI of 40:60 silage:concentrate. In both Exp. 1 and 2, experimental periods consisted of 28 d, incorporating a 13-d acclimatization, a 5-d measurement period, and a 10-d washout period. In Exp. 1, enteric CH(4), feed apparent digestibility, and feed intake were measured over the 5-d measurement period. In Exp. 2, rumen fluid was collected on d 16 to 18, immediately before (a.m.) feeding and 2, 4, 6, and 8 h thereafter. Rumen pH was determined and samples were taken for protozoa count, VFA, and ammonia analysis. Enteric CH(4) emissions were estimated by using the sulfur hexafluoride tracer technique and feed apparent digestibility was estimated by using chromic oxide as an external marker for fecal output. In Exp. 1, increasing dietary MA led to a linear decrease in total DMI (P < 0.001) and total daily CH(4) emissions (P < 0.001). Compared with the control diet, the greatest concentration of MA decreased total daily CH(4) emissions by 16%, which corresponded to a 9% reduction per unit of DMI. Similarly, in Exp. 2, inclusion of MA reduced DMI in a linear (P = 0.002) and quadratic (P < 0.001) fashion. Increasing dietary MA led to a linear decrease in molar proportion of acetic (P = 0.004) and butyric acids (P < 0.001) and an increase in propionic acid (P < 0.001). Ruminal pH tended to increase (P = 0.10) with increasing dietary MA. Dietary inclusion of MA led to a linear (P = 0.01) decrease in protozoa numbers. Increasing supplementation with MA decreased CH(4) emissions, but DMI was also decreased, which could have potentially negative effects on animal performance.

Embryo yield and quality following dietary supplementation of beef heifers with n-3 polyunsaturated fatty acids (PUFA).

The objective of this study was to examine the effects of dietary n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation on embryo yield and quality in heifers. Animals were individually offered barley straw and concentrate diets supplemented with either palmitic acid (C16:0; CON) or a partially rumen protected n-3 PUFA-enriched supplement. Following oestrous cycle synchronisation, superovulation was induced using FSH. Blood samples were collected for the measurement of fatty acids, metabolites, insulin and IGF-1. On day 7 post-insemination the number of ovulations was estimated and embryos recovered non-surgically and quality graded. At embryo recovery 50 ml of the uterine flushing was collected from each horn for fatty acid analysis. Grade 1 embryos were isolated, snap frozen in liquid nitrogen and stored at -80 degrees C. mRNA expression for six genes, LIF, BAX, Cx43 and E-CAD associated with embryo development, and PPAR-alpha and -delta, associated with lipid metabolism was analysed. The n-3 PUFA supplementation increased plasma n-3 PUFA concentration (P<0.05) and reduced n-6:n-3 PUFA ratio (P<0.05). Uterine concentration of the n-3 PUFA, eicosapentaenoic acid was increased (P<0.05) and the concentration of arachidonic acid decreased (P<0.05) following n-3 PUFA supplementation. While CON increased triglyceride concentrations, diet did not affect the other plasma metabolites, insulin or IGF-1 (P>0.05). Similarly, there was no effect of diet on superovulation rate, embryo recovery rate, embryo quality (P>0.05) or mRNA expression of the genes examined (P>0.05).

Effect of level of dietary n-3 polyunsaturated fatty acid supplementation on systemic and tissue fatty acid concentrations and on selected reproductive variables in cattle.

Reproductively normal crossbred beef heifers were individually offered a diet of barley straw and concentrate supplemented with one of four levels of a fish oil (FO) enriched supplement. Following oestrous cycle synchronisation, blood samples were collected at appropriate intervals for the measurement of progesterone (P(4)), oestradiol (E(2)), fatty acids, insulin-like growth factor 1 (IGF-1) and metabolites. On days 15 and 16 of the cycle, oxytocin was administered intravenously and the prostaglandin F(2alpha) (PGF(2alpha)) response was measured as venous concentrations of 13,14-dihydro-15-keto PGF(2alpha) (PGFM). The heifers were slaughtered on days 17 or 18 of the oestrous cycle and endometrial tissue, rumen fluid and follicular fluid were collected for determination of fatty acid concentrations. In general there was no effect (P>0.05) of diet on plasma P(4) or E(2) concentrations. Increasing FO supplementation increased CL diameter on day 7 post-oestrus (P<0.0001) but had no effect on diameter on day of slaughter (P>0.05). On day 15, PGFM concentration was greater on the highest level of FO supplementation compared to controls (P<0.05), however, there were no differences between other diet comparisons (P>0.05). There was no effect of diet on PGFM concentration on day 16 (P>0.05). There was a strong positive relationship between plasma and uterine endometrial concentrations of both EPA (R(2)=0.86; P<0.0001) and total n-3 PUFA (R(2)=0.77; P<0.0001). IGF-1 concentrations increased on all diets and were greatest at the highest level of n-3 PUFA supplementation (P<0.05).

Effect of dietary enrichment with either n-3 or n-6 fatty acids on systemic metabolite and hormone concentration and ovarian function in heifers.

The objective of this experiment was to examine the effects of dietary n-3 or n-6 fatty acid (FA) supplementation on blood FA, metabolite and hormone concentrations, follicle size and dynamics and corpus luteum (CL) size. Reproductively normal heifers (n = 24) were individually fed diets of chopped straw and concentrate containing either (i) no added lipid (CON; n = 8); (ii) 2% added fat as whole raw soya beans (WSB, n-6; n = 8); or (iii) 2% added fat as fish oil (FO, n-3; n = 8). Following oestrous cycle synchronisation, blood samples were collected at appropriate times and intervals for the measurement of hormones, FAs and metabolites. On days 15 and 16 of the cycle, animals were subjected to an intravenous oxytocin challenge and prostaglandin F2α (PGF2α) response, measured as venous concentrations of 13,14-dihydro-15-keto PGF2α (PGFM). Dry matter intake and average daily gain were similar among treatments (P > 0.05). Plasma concentration of linoleic acid was highest on WSB (P < 0.05), while eicosapentaenoic (EPA, n-3; P < 0.0001) and docosahexaenoic acid (DHA, n-3; P < 0.0001) were greatest in the FO group. Plasma concentrations of arachidonic acid were higher on FO (P < 0.05) compared with CON and WSB. Plasma triglyceride concentrations increased, while ß-hydroxybutyrate (BHBA) decreased with time on all diets (P < 0.05). There was a diet × time interaction (P < 0.01) for non-esterified fatty acid (NEFA) concentrations. Plasma cholesterol was higher on WSB and FO (P < 0.01) compared with CON. Progesterone (P4) and oestradiol (E2) concentrations, as well as follicle growth rate and CL diameter were similar across diets (P > 0.05). There was a diet × day interaction for PGFM (P < 0.01). When corrected for systemic E2 : P4 ratio, day 15 concentrations of PGFM were higher in the WSB group at 15 and 30 min (P < 0.01) post oxytocin administration compared with CON and FO, which were similar (P > 0.05). Concentrations of PGFM on day 16 were similar for WSB and FO and were greater than CON at 15 (P < 0.01) and 45 min (P < 0.05) post oxytocin administration, and at 30 min for FO (P < 0.05). With the exception of PGFM, dietary lipid source did not affect the reproductive variables measured.

Expert judgments of hypnosis from subjective state reports.

Suggestibility was assessed in 60 college students after a traditional hypnotic induction, an alert induction, progressive relaxation training, or instruction in goal-directed imagery. Responsiveness to suggestion did not differ between groups. Subjects also generated open-ended reports of their states of awareness and of their experience of 3 hypnotic suggestions. A sample of these reports from 24 moderately to highly suggestible subjects were evaluated by 18 experts in the field of hypnosis. Expert ratings of subjects' open-ended reports indicated that (a) traditional hypnotic inductions produce a state of consciousness that is indistinguishable from nonhypnotic relaxation training, (b) the subjective experience of hypnotic suggestions after imagination training is indistinguishable from that after hypnotic inductions, and (c) suggestibility is unrelated to state of consciousness as assessed by experts.