CSIRO PUBLISHING
Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD14348
Lactation-induced changes in metabolic status and follicular-fluid metabolomic profile in postpartum dairy cows Niamh Forde A,D, Aoife O’Gorman A,B, Helena Whelan A,B, Pat Duffy A, Lydia O’Hara A, Alan K. Kelly A, Vitezslav Havlicek C, Urban Besenfelder C, Lorraine Brennan A,B and Pat Lonergan A A
School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland. Institute of Food and Health, University College Dublin, Belfield, Dublin 4, Ireland. C Reproduction Centre – Wieselburg, University of Veterinary Medicine Vienna, Veterina¨rplatz 1, A-1210, Austria. D Corresponding author. Email:
[email protected] B
Abstract. The aim was to investigate the effect of lactation on the composition of pre-ovulatory follicular fluid (FF). Forty in-calf primiparous heifers and 20 maiden heifers were enrolled. Immediately after calving, half of the cows were dried off while the remainder were milked twice daily. Serum samples were collected twice weekly from two weeks pre- to 84 days postpartum (dpp). FF was analysed by gas chromatography–mass spectrometry. Serum concentrations of non-esterified fatty acids and b-hydroxybutyrate were higher, while glucose, insulin and Insulin-like growth factor 1 (IGF1) concentrations were lower in lactating cows compared with non-lactating cows and heifers (P , 0.01). Principal component analysis of FF metabolites revealed a clear separation of the lactating group from both non-lactating cows and heifers. The amino acids tyrosine, phenylalanine and valine and fatty acids heneicosanoic acid and docosahexaenoic acid were all lower in FF from lactating compared with dry cows (P , 0.05). FF from lactating cows was higher in aminoadipic acid, a-aminobutyric acid, glycine and serine while histidine, leucine, lysine, methionine and ornithine were all lower than in dry cows and heifers (P , 0.05). The ratio of n6 : n3 was higher in lactating cows compared with both non-lactating cows and heifers, whereas total n3 polyunsaturated fatty acids, pentadecanoic, linolenic, elaidic and arachidonic acids were all lower in the FF of lactating cows than both non-lactating cows and heifers (P , 0.05). In conclusion, lactation induces distinct changes in the overall metabolic status of postpartum lactating dairy cows which are associated with divergent metabolite profiles in FF. Additional keywords: amino acids, diary heifers, fatty acids, non-lactating dairy cows, pre-ovulatory follicle.
Received 17 September 2014, accepted 14 May 2015, published online 15 June 2015
Introduction The physiological changes associated with milk production impact on circulating metabolites during the early postpartum period and are likely to play a role in poor reproductive efficiency in high-producing dairy cows. Most transition cows enter a state of negative energy balance (NEB) due to increased energy demands at parturition, decreased dry matter intake (DMI) shortly before parturition and a consequent deficit between DMI and the energy demands associated with milk production. This deficit is met by mobilising adipose tissue in the form of non-esterified fatty acids (NEFA). Thus, greater blood concentrations of NEFA and lower blood concentrations of glucose are indicative of the normal process of nutrient partitioning that occurs early postpartum in dairy cows. However, prolonged elevated concentrations of NEFA and associated Journal compilation Ó CSIRO 2015
b hydroxybutyrate (BHB) have a detrimental effect on reproduction and increase the risk of periparturient clinical disease (Ospina et al. 2010a, 2010b, 2010c; Garverick et al. 2013). While the causes of infertility in dairy cows are almost certainly multifactorial, the quality of the oocyte is central to a successful outcome. Evidence pointing towards a contribution of poor oocyte quality to infertility in postpartum dairy cows comes from a variety of sources. For example, data from nonsurgical uterine flushing of lactating dairy cows (reviewed by Sartori et al. 2010) suggest that up to 50% of embryos fail to survive to Day 6 or 7 (Cerri et al. 2009a, 2009b, 2009c). Furthermore, higher pregnancy rates have been reported in lactating dairy cows after embryo transfer, which bypasses the cow’s own oocyte, compared with artificial insemination (AI; Putney et al. 1989; Ambrose et al. 1999; Drost et al. 1999; www.publish.csiro.au/journals/rfd
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Rutledge 2001; Al-Katanani et al. 2002; Vasconcelos et al. 2006; Demetrio et al. 2007). Follicles that are growing shortly after calving reach ovulatory size ,60 to 90 days postpartum (approximate time of first insemination). Thus, the follicle and the oocyte within may be compromised in postpartum cows due to extreme concentrations of circulating hormones and metabolites (Britt 1992; Leroy et al. 2004b). In support of this hypothesis, exposure of oocytes in vitro to NEFA at concentrations consistent with those found in the pre-ovulatory follicle of postpartum lactating cows is detrimental to oocyte development (Leroy et al. 2004a, 2005). Detailed studies on the composition of bovine follicular fluid, and in particular how this composition is affected by lactation, are scarce. This is surprising given the importance of the follicular environment in determining oocyte quality (Rizos et al. 2002). Bender et al. (2010) reported significant differences in the concentration of 24 fatty acids and nine aqueous metabolites in pre-ovulatory follicular fluid of postpartum dairy cows compared with that of nulliparous heifers. While interesting, the model used is somewhat confounded by parity status. More recently, Matoba et al. (2014) reported differences in the aqueous metabolites in follicular fluid between oocytes that formed blastocysts after IVF and those that degenerated; L-alanine, glycine and L-glutamate were positively correlated and urea was negatively correlated with blastocyst formation. Follicular fluid associated with competent oocytes was significantly lower in palmitic acid and total fatty acids and significantly higher in linolenic acid than follicular fluid from incompetent oocytes. Here, we tested the hypothesis that the systemic insult of NEB in dairy cows results in a compromised follicular-fluid microenvironment. To test this hypothesis we characterised the metabolic status of age-matched postpartum primiparous dairy cows that were either milked after calving (i.e. lactating) or were dried off immediately at calving (i.e. never milked, nonlactating) as well as a group of non-pregnant maiden heifers. In a subset of animals from each group, we analysed the metabolic composition of follicular fluid from the pre-ovulatory dominant follicle (35–45 days postpartum) in order to better understand the factors contributing to poor oocyte quality in lactating cows. Materials and methods Animal model All experimental procedures involving animals were licenced by the Department of Health and Children, Ireland, in accordance with the Cruelty to Animals Act, 1876, and the European Community Directive 86/609/EC. All procedures were sanctioned by University College Dublin’s Animals Research Ethics Committee. Forty in-calf primiparous Holstein–Friesian heifers and 20 non-pregnant Holstein–Friesian heifers with a similar economic breeding index were enrolled into the study. At calving, cows were randomly assigned to one of two groups: (1) lactating (n ¼ 20) or (2) non-lactating (n ¼ 20). From calving, animals in the lactating group were milked twice per day (0700 and 1600 hours), while those in the non-lactating group were dried off immediately after calving (i.e. never milked) as previously
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described (Maillo et al. 2012). Prior to calving, all animals were fed 30 kg grass silage per head per day with pregnant heifers getting an additional 3 kg concentrates per head per day. After calving, non-lactating cows received ad libitum grass silage plus 4 kg concentrates per day, while lactating cows received 24 kg maize silage per 16 kg grass silage plus 7 kg concentrates. Beginning 2 weeks before expected calving date, all animals were weighed, body condition score (BCS) recorded and blood sampled twice weekly until Day 85 postpartum. Similar measurements were recorded for the non-pregnant heifers over the same period. Blood samples were taken from the jugular vein into either plain redtop vacutainer tubes (for serum collection) or fluoride oxylate-coated vacutainers (for glucose analysis). All samples were stored at 48C overnight, centrifuged at 1500g for 20 min at 48C and decanted. Samples were then stored at 208C before analysis. Analysis of metabolic status Circulating concentrations of metabolites were analysed as previously described (Maillo et al. 2012). Insulin concentrations in serum were measured using a solid-phase 125I radioimmunoassay, Insulin Coat-A-Count kit (Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA) with a sensitivity of 1.2 mIU mL1. The inter- and intra-assay coefficients of variation (CV) for insulin were 19.0% and 15.1%, 9.8% and 12.3% and 2.8% and 8.8% for the low, medium and high quality controls (QC), respectively. Serum insulin-like growth factor 1 (IGF1) concentrations were measured by radioimmunoassay. The interassay CV were 13.6%, 6.1% and 4.9% and the intra-assay CV were 13.6%, 12.2% and 13.0% for the low, medium and high QCs, respectively. Serum BHB concentrations were measured using RANBUT D-3-hydroxybutyrate (Randox Laboratories Ltd, Crumlin, UK) using a kinetic enzymatic reaction with a sensitivity of 0.1 nmol L1. The intra-assay CV were 3.2% and 1.82% for the low and high QCs, respectively. Circulating NEFA concentrations were measured using the Randox NEFA enzyme assay with a sensitivity of 0.072 mmol L1 with intra-assay CV of 1.05 and 3.4% for the medium and high QCs, respectively. Plasma glucose concentrations were measured using the automated Randox glucose hexokinase (Gluc-HK) enzymatic method (sensitivity 0.662 mmol L1). The intra-assay CV for the medium and high quality control standards were 1.3 and 1.7%, respectively. Follicular-fluid recovery Follicular fluid (FF) from the pre-ovulatory dominant follicle was recovered via transvaginal endoscopic follicle aspiration. Approximately 35–45 days postpartum (dpp) the oestrous cycles of all animals (lactating and non-lactating postpartum cows and maiden heifers) were synchronised. A controlled intravaginal drug device (CIDR; Pfizer Animal Health, Sandwich, UK) containing 1.38 g of progesterone (P4) was inserted for 8 days. One day before CIDR removal each animal received a 2-mL intramuscular (i.m.) injection of a prostaglandin F2a analogue (PG, Estrumate; Intervet, Dublin, Ireland; equivalent to 0.5 mg Cloprostenol) to regress the endogenous corpus luteum (CL). Thirty-six hours after CIDR removal each animal
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received a 2.5 mL i.m. injection of Receptal, equivalent to 0.012 mg buserelin (Intervet) and follicle aspiration was performed, on average, 22 h later. Prior to follicle aspiration, each animal was scanned for the presence of a pre-ovulatory follicle, which was measured. All animals received 5 mL epidural anaesthesia (20 mg mL1 lidocaine hydrochloride, Norocaine; Norbrook Laboratories Ltd, Newry, Northern Ireland). A trocar was inserted via the vagina into the peritoneal cavity in the mid-dorsal area close to the portio of the cervix, which served for the introduction of a 308 oblique endoscope (Maillo et al. 2012). Once the preovulatory follicle was located, an 18-gauge needle was pushed through the follicular wall and the follicular fluid was aspirated into individual collecting tubes. The volume of FF recovered was recorded and it was then centrifuged at 100g for 5 min at room temperature to pellet any cellular debris. The supernatant was removed into a new tube, snap frozen in liquid nitrogen and stored at 808C before analysis. Analysis of follicular-fluid metabolites The follicular fluid was analysed for both amino-acid and fattyacid metabolites using gas chromatography–mass spectrometry (GC/MS). Follicular fluid from heifers (n ¼ 6), non-lactating (n ¼ 5) and lactating cows (n ¼ 6) was thawed on ice before extraction. The Phenomenex EZ:faast kit (Phenomenex, Cheshire, United Kingdom) was used for amino-acid analysis. Briefly, 100 mL of follicular-fluid sample was combined with 100 mL of 0.2 nM Norvaline as an internal standard. A solidphase extraction step was performed followed by derivatisation of the extracted amino acids to form chloroformates. This was followed by a liquid–liquid extraction, with the upper layer removed and dried under nitrogen. Samples were re-dissolved in 100 mL of a mixture of iso-octane and chloroform (80 : 20) and analysed by GC/MS. The GC/MS system comprised of an Agilent 7890A GC (Agilent Technologies GMBH, Waldbronn, Germany) coupled with a 5975C ion-trap MS (Agilent Technologies GMBH) running in electron ionisation running in positive mode (EIþ) mode. Chromatography was performed on a Zebron ZB-AAA capillary column (10 m 0.25 mm; Phenomenex, Cheshire, UK) using helium at 1.1 mL min1. Samples (1 mL) were injected into a programmed temperature ramp using a split mode. The GC temperature was initially 1108C for 2 min and increased to 3208C at 308C min1. After a solvent delay of 1 min, mass spectra were recorded within a scan range of 35–550 atomic mass units (amu) at an electron energy of 70 eV and a source temperature of 2308C. The organic component of the follicular fluid was analysed by combining 200 mL of follicular fluid with 20 mL of 2 mg mL1 nonadecanoic acid (19 : 0) as an internal standard and extracted using a 1 : 2 mixture of chloroform : methanol. Extracts were derivatised by methylation using methanolic BF3 and derivatives were re-suspended in 200 mL hexane and analysed on an Agilent 7890A GC coupled with a 5975C MS with an Agilent HP-5 MS column (30 m 250 mm 0.25 mm; Agilent Technologies GMBH). One microlitre of the derivatised sample was injected in splitless mode and the initial oven temperature of 70 8C was raised to 220 8C at 5 8C min1, held
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for 20 min and then raised to 320 8C at 20 8C min1. Calibration was achieved by comparison of peak areas for amino acids and fatty acids with reference to known standards (EZ:fast kit standards; Phenomenex and Supelco 37 component FAME mix; Sigma Aldrich, Ireland) using Agilent Chemstation (software version MSD E.02.00.493) and by comparison of their mass spectra with those in the NIST Library 2.0. Automatic peak detection was carried out with Agilent Chemstation MSD (Agilent Technologies GMBH). Mass spectra deconvolution was performed with the Automated Mass Spectral Deconvolution and Identification System (AMDIS, Version 2.65; http:// www.amdis.net/). Peaks with a signal-to-noise ratio of lower than 30, which is an acceptable level to avoid false positives, were rejected (Norli et al. 2010). To obtain accurate peak areas for the internal standard and specific peaks or compounds, one quant mass for each peak was specified as the target ion and three masses were selected as qualifier ions. Each data file was manually analysed for false positives and negatives in Agilent Chemstation. Concentrations reported for amino acids and fatty acids are expressed a percentages (%) s.e.m. of the total concentrations. Total saturated fatty acid (SFA) fraction, total monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) fractions, indices of desaturase enzyme activity in C16 fatty acids (D9-desat (16)) and in C18 fatty acids (D9-desat (18)) and elongase enzyme activity in chain lengthening of C16–C18 fatty acids were calculated using previously published equations (Malau-Aduli et al. 1998). Statistical analysis All data (weight, BCS and circulating concentrations of NEFA, BHB, insulin, IGF1 and glucose) were checked for normality and homogeneity of variance by histograms, quantile-quantile (qq) plots and formal statistical tests as part of the UNIVARIATE procedure of SAS (Version 9.1.3; SAS Institute, Cary, NC, USA). Data that were not normally distributed were transformed by raising the variable to the power of lambda. The appropriate lambda value was obtained by conducting a Box–Cox transformation analysis using the TRANSREG procedure of SAS. The transformed data were used to calculate P values. The corresponding least-squares means and standard error of the non-transformed data are presented in the results for clarity. Concentrations of circulating metabolites as well as weight and BCS were analysed using repeated-measures with the MIXED procedure of SAS. Fixed effects included experimental treatment (heifer, lactating or non-lactating), day and their interactions. The interaction term, if not statistically significant (P . 0.10), was subsequently excluded from the final model. Animal-within-treatment was included as a random effect. The type of variance–covariance structure used was chosen depending on the magnitude of the Akaike information criterion for models run under compound symmetry, unstructured, autoregressive or Toeplitz variance–covariance structures. Differences between treatments were determined by F-tests using Type III sums of squares. The PDIFF command incorporating the Tukey test was applied to evaluate pairwise comparisons between treatment means. Pearson correlation
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coefficients between systemic metabolic traits and amino-acid and fatty-acid components of the follicular fluid were generated using the CORR procedure of SAS. Significant strong (r . 0.6), moderate (r . 4 and , 6) or weak (r , 0.04) correlations were identified when P , 0.05. Principal component analysis (PCA) was performed on both the amino-acid and fatty-acid follicularfluid datasets using SIMCA-Pþ11 (Umetrics, Crewe, UK). PCA was used to identify separation between the study groups and outliers as appropriate. Correlation analysis, general linearmodel analysis and post hoc Bonferroni’s test were performed using SPSS 14 (SPSS Inc., Chicago, IL, USA). Results Characterisation of the metabolic status of lactating and non-lactating cows and maiden heifers In order to assess the metabolic status of the lactating and nonlactating cows and maiden heifers, all data were analysed in two time periods, with Period 1 being before parturition (for the nonlactating and lactating cows) or the first 2 weeks on the trial (heifers), and all other analyses were performed on samples or measurements taken after parturition (non-lactating and lactating cows) or from 2 weeks after enrolment in the trial for the heifer group. The bodyweight and BCS of non-lactating and lactating cows were significantly lower than nulliparous heifers (P , 0.0001; Fig. 1a, b). A significant day*treatment interaction was observed for both parameters (P , 0.0001) caused by an inversion of profiles for non-lactating and lactating cows between Day 28 and Day 35 postpartum . Heifers had significantly higher serum insulin concentrations in the pre-partum period and up to 10 dpp than both postpartum groups. Lactating cows had basal insulin concentrations (,3 mIU mL1) throughout the study period (P , 0.0001) while in the non-lactating postpartum cows, insulin concentrations were similar to those in maiden heifers from ,10 dpp (Fig. 1c). Circulating IGF1 concentrations were higher in heifers than both postpartum groups throughout the period of study (P , 0.0001). IGF1 concentrations decreased in the 2 weeks before calving and remained low in postpartum lactating cows, while in non-lactating cows concentrations increased gradually from calving to Day 84 postpartum (Fig. 1d). Pre-partum glucose concentrations were lower in pregnant cows than non-pregnant heifers (P , 0.0001). Glucose concentrations peaked at calving in all pregnant cows. After calving, glucose concentrations were similar in non-lactating cows and heifers and were significantly lower (P , 0.0001) in lactating cows from Day 7 postpartum onwards (Fig. 1e). Concentrations of NEFA were basal (,0.2 mmol L1) in maiden heifers throughout the study. Both lactating and non-lactating postpartum cows exhibited an elevation in NEFA beginning 10 days before calving. In the non-lactating group, NEFA concentrations returned to basal levels rapidly after calving (by 10 dpp), whereas in lactating cows concentrations were significantly elevated until approximately Day 42 (Fig. 1f ). Similarly, BHB concentrations were basal in maiden heifers throughout the study period. Lactation significantly increased BHB concentrations postpartum; concentrations returned to basal levels by 10 dpp in non-lactating cows but remained significantly elevated in lactating cows (P , 0.0001; Fig. 1g).
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Amino-acid metabolites in follicular fluid The metabolomic profile of follicular fluid from non-lactating cows (n ¼ 5), lactating cows (n ¼ 6) and heifers (n ¼ 6) was analysed. Mean follicle diameter (s.e.m.) was 14.6 (1.34), 16.53 (0.83) and 17.04 (1.22) mm for the non-lactating cows, lactating cows and maiden heifers, respectively. PCA of the amino-acid profiles revealed clear separation of the lactating cows from the non-lactating cows and heifer group (Fig. 2a). Of the 22 amino-acid metabolites identified in the follicular fluid of the pre-ovulatory dominant follicle, the most abundant were the amino acids glycine, alanine and glutamine (Table 1). There was no treatment effect on the abundance of alanine, isoleucine, threonine, asparagine, aspartic acid, hydroxyproline, glutamic acid, glutamine or tryptophan (P . 0.05). The concentration of proline was higher in the lactating cows compared with heifers (P , 0.05), whereas tyrosine, phenylalanine and valine were all higher in the follicular fluid of non-lactating compared with lactating cows only (P , 0.05). Follicular fluid from the pre-ovulatory follicle of lactating cows was higher in a-aminoadipic acid (AAA), a-aminobutyric acid (ABA), glycine and serine metabolites, whereas histidine, leucine, lysine, methionine and ornithine were all lower in the follicular fluid of lactating cows compared with both the non-lactating cows and maiden heifers (P , 0.05). Fatty-acid metabolites in follicular fluid Of the 23 fatty acids detected in the pre-ovulatory follicular fluid, the abundance of 13 was significantly altered by the metabolic status of the animal (or their relative ratios: Table 2). Similar to the amino-acid profile, the overall fatty-acid profile (depicted by PCA analysis) was significantly different in lactating cows compared with their non-lactating and heifer counterparts (Fig. 2b). The concentration of cis-10-heptadecanoic, heptadecanoic and cis-5, 8, 11, 14, 17-eicosapentaenoic acid (EPA) were all lower, whereas myristic and linoleic acids were higher in follicular fluid of lactating cows compared with maiden heifers (P , 0.05). Heneicosanoic acid and cis-4, 7, 10, 13, 16, 19docosahexaenoic acid (DHA) were both lower in the follicular fluid of lactating compared with non-lactating cows (P , 0.05). The ratio of n6 : n3 was significantly higher in lactating cows compared with both non-lactating cows and heifers, while total n3 PUFAs, pentadecanoic, linolenic, elaidic and arachidonic acids were all significantly lower in the follicular fluid of lactating cows compared with both the non-lactating and maiden heifer groups (P , 0.05). Correlation analysis of systemic concentrations of metabolic status and composition of follicular fluid Analysis of the correlation between systemic markers of energy balance and the amino-acid composition of follicular fluid are given in Table 3. AAA concentrations in follicular fluid were negatively correlated with circulating concentrations of IGF1 (0.67941) and insulin (0.54412), whereas AAA in follicular fluid correlated positively with concentrations of BHB (0.60562) and NEFA (0.55392) in circulation. ABA concentrations in follicular fluid were negatively correlated with IGF1 (0.54118), insulin (0.69412) and glucose (0.62653) in circulation but there was a positive correlation between BHB
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(b)
Weight (kg)
600
Post calving: Day P 0.001 Treatment P 0.0001 ∗ Day Treatment P 0.0001
4.00
Body condition score (AU)
(a)
550
500
3.50
3.00 2.75 2.50
9.5
( ) ( 14 ) 1 dpp ( 0 d ) pp ( 7 d ) 3 pp dp 3 p dp 7 p d 10 pp d 14 pp d 21 pp d 28 pp d 35 pp d 42 pp d 49 pp d 56 pp d 63 pp d 70 pp d 77 pp 84 dpp dp p
Post calving: Day P 0.0001 Treatment P 0.05 ∗ Day Treatment P 0.05
7.5 5.5 3.5
(d)
500
IGF1 concentrations in serum (ng mL1)
Insulin concentrations in serum (μlU mL1)
(c) 11.5
450
350 300 250 200 150 100 50
(
)1 ( 4 d ) 1 pp ( 0 d ) 7 pp ( dp )3 p C dpp al vi n 3 g dp 7 p dp 10 p d 14 pp d 21 pp d 28 pp d 35 pp d 42 pp d 49 pp d 56 pp dp 63 p d 70 pp d 77 pp d 84 pp dp p
( ) ( 14 ) 1 dp ( 0 d p ) pp ( 7d ) 3 pp C dp al p vi n 3 g dp 7 p d 10 pp d 14 pp d 21 pp d 28 pp 35 dpp d 42 pp d 49 pp 56 dpp d 63 pp d 70 pp d 77 pp 84 dpp dp p
NEFA concentrations in serum (mmol L1)
4.3
(f )
Post calving: Day P 0.0001 Treatment P 0.0001 ∗ Day Treatment P 0.0001
4.1 3.9 3.7 3.5 3.3
0.8
Post calving: Day P 0.0001 Treatment P 0.0001 ∗ Day Treatment P 0.0001
Post calving: Day P 0.1 Treatment P 0.0001 Day∗Treatment P 0.0001
0.6
0.4
0.2
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)1 ( 4 d ) 1 pp 0 ( dp )7 p ( dp )3 p d C pp al vi n 3 g dp p 7 dp 10 p dp 14 p dp 21 p dp 28 p dp 35 p dp 42 p dp 49 p dp 56 p dp 63 p dp 70 p dp 77 p dp 84 p dp p
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(g)
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
( ) ( 14 ) 1 dp p ( 0 d ) p ( 7d p ) 3 pp C dpp al vi n 3 g dp 7 p d 10 pp d 14 pp d 21 pp d 28 pp d 35 pp 42 dpp d 49 pp dp 56 p d 63 pp 70 dpp d 77 pp d 84 pp dp p
( ) ( 14 ) 1 dp p ( 0 d ) pp ( 7 d ) 3 pp d C pp al vi n 3 g dp 7 p d 10 pp d 14 pp d 21 pp d 28 pp d 35 pp d 42 pp d 49 pp d 56 pp d 63 pp d 70 pp dp 77 p d 84 pp dp p
Glucose concentrations in serum (mmol L1)
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Post calving: Day P 0.0001 Treatment P 0.0001 ∗ Day Treatment P 0.0001
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Post calving: Day P 0.0001 Treatment P 0.0001 ∗ Day Treatment P 0.0001
3.25
( ) ( 14 ) 1 dpp ( 0 d ) pp ( 7 d ) 3 pp dp 3 p dp 7 p d 10 pp d 14 pp d 21 pp dp 28 p d 35 pp d 42 pp dp 49 p d 56 pp d 63 pp d 70 pp d 77 pp d 84 pp dp p
450
3.75
Days post partum (dpp)
Fig. 1. Average ( s.e.m.) weekly (a) bodyweight (kg), (b) body condition score (BCS) and circulating concentrations of (c) insulin (mIU mL1), (d ) IGF1 (ng mL1), (e) glucose (mmol L1), ( f ) NEFA (mmol L1) and (g) BHB (mmol L1) for heifers (diamond), non-lactating cows (squares) and lactating cows (triangle). Samples were taken twice a week for 2 weeks prior to parturition and 2 weeks postpartum and once per week thereafter. Samples for heifers were taken twice a week for the first 4 weeks of being enrolled in the trial and then once a week thereafter until slaughter. P-values are given for the effects of Day, Treatment and their interactions and were considered significant when P , 0.05.
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t[1] Fig. 2. Principal component analysis (PCA) score plot of PC1 vs PC2 for (a) amino-acid and (b) fatty-acid metabolite profiles from the follicular fluid of heifers, lactating and non-lactating cows. (a) R2 ¼ 0.73 for a two-component model; data was pareto scaled. (b) R2 ¼ 0.72 for a two-component model; data was unit-variance scaled.
Table 1. Amino-acid metabolites identified in the follicular fluid (FF) of the pre-ovulatory dominant follicle from dairy heifers (n 5 6), non-lactating (n 5 5) and lactating (n 5 6) dairy cows (35–45 days postpartum) Values are expressed as percentages (%) of the total concentration of amino acids identified in follicular fluid from this cohort of animals. AAA, a-aminoadipic acid; ABA, a-aminobutyric acid. Significant differences in metabolite abundance are denoted by different superscripts when P , 0.05: asignificantly different from the heifer group, bsignificantly different from non-lactating cows, csignificantly different from lactating cows; ns, not significant Amino acid
Heifers (n ¼ 6)
Non-lactating (n ¼ 5)
Lactating (n ¼ 6)
P value
AAA ABA Alanine Asparagine Aspartic acid Glutamic acid Glutamine Glycine Histidine Hydroxyproline Isoleucine Leucine Lysine Methionine Ornithine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
0.04 0.01 0.84 0.31c 12.75 3.57 1.79 0.37 0.41 0.08 4.97 2.09 10.99 5.37 19.08 3.01c 2.51 0.74c 1.09 0.42 4.39 0.58 6.61 0.91c 5.71 0.82c 1.4 0.35c 3.1 0.82c 3.34 0.59 2.91 0.38c 2.67 0.82c 2.99 0.9 1.61 0.46 2.51 0.5 8.29 1.65
0.05 0.01 0.85 0.2 c 11.67 2.61 1.79 0.27 0.44 0.24 3.79 1.74 12.27 3.49 15.87 2.11c 2.49 0.51c 1.0 0.43 4.71 0.21 7.15 0.42c 6.04 1.21c 1.54 0.18c 2.93 0.81c 3.86 0.42c 3.15 0.34 2.37 0.483 3.84 0.41 1.77 0.32 3.32 0.8c 9.07 0.92c
0.09 0.03 2.19 0.48a,b 15.13 3.37 2.15 0.27 0.31 0.09 3.45 1.08 8.15 3.13 27.34 3.16a,b 1.31 0.79a,b 0.74 0.23 4.29 0.53 4.34 0.9a,b 3.94 1.13a,b 0.92 0.27a,b 1.1 0.13a,b 2.65 0.45b 3.84 0.77a 4.29 1.43a,b 3.14 0.83 1.42 0.21 2.17 0.6b 7.03 0.48b
1.1 103 1.3 205 ns ns ns ns ns 2.7 105 1.6 102 ns ns 8.2 105 1.1 102 5.5 103 2.0 104 4.5 103 2.7 102 1.4 102 ns ns 2.8 102 3.0 102
c
in circulation and ABA in follicular fluid (0.52585). Aspartic acid was strongly correlated with glucose in circulation (0.71434) but was not significantly correlated with any of the other measured metabolites, whereas leucine concentrations in follicular fluid were positively correlated with both insulin (0.57353) and glucose (0.71285) concentrations but negatively associated with BHB in circulation (0.74299). Both methionine and valine
c
a,b
concentrations in follicular fluid were negatively correlated with the systemic metabolite BHB (–0.51699 and 0.55835, respectively). Ornithine was positively correlated to circulating concentrations of IGF1 (0.65294) and insulin (0.5) but negatively correlated with BHB (0.61891) in circulation. Phenylalanine was positively correlated with glucose (0.5923) but negatively correlated with BHB concentrations in circulation (0.613),
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Table 2. Fatty-acid metabolites identified in the follicular fluid (FF) of the pre-ovulatory dominant follicle from dairy heifers (n 5 6), non-lactating (n 5 5) and lactating (n 5 6) dairy cows (35–45 days postpartum) Values are expressed as percentages (%) of the total concentration of fatty-acid metabolites identified in follicular fluid from this cohort of animals. EPA, cis-5, 8, 11, 14, 17-eicosapentaenoic acid; DHA, cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid. Significant differences in metabolite abundance are denoted by different superscripts when P , 0.05: 1significantly different from the heifer group, 2significantly different from non-lactating cows, 3significantly different from lactating cows Fatty acid Myristic Pentadecanoic Palmitic Palmitoleic Heptadecanoic Cis-10-Heptadecanoic Stearic Oleic Elaidic Linoleic Linolenic g-Linolenic Arachidic cis-11,14-Eicosadienoic cis-8,11,14-Eicosatrienoic Arachidonic EPA Heneicosanoic Behenic DHA Tricosanoic Lignoceric Nervonic Total SFA Total MUFA Total PUFA n3 PUFA n6 PUFA n6 : n3
14 : 0 15 : 0 16 : 0 16 : 1 17 : 0 17 : 1 18 : 0 18 : 1n9c 18 : 1n9t 18 : 2n6c 18 : 3n3 18 : 3n6 20 : 0 20 : 2 20 : 3n6 20 : 4n6 20 : 5n3 21 : 0 22 : 0 22 : 6n3 23 : 0 24 : 0 24 : 1
Heifers (n ¼ 6)
Non-lactating (n ¼ 5)
Lactating (n ¼ 6)
P value
0.40 0.07c 0.91 0.23c 13.29 1.05 1.98 0.21 0.87 0.22c 1.05 0.08c 13.00 2.08 15.25 1.68 1.66 0.47c 33.69 5.16c 4.43 1.18c 0.53 0.11 0.06 0.01 0.13 0.02 1.95 0.52 5.19 1.04c 3.74 1.10c 0.02 0.00 0.19 0.04 0.68 0.18 0.36 0.07 0.32 0.06 0.30 0.05b 29.41 1.81 20.24 1.43 50.23 3.14 8.84 1.97c 41.36 4.05 5.05 2.11c
0.48 0.16 1.00 0.19c 13.39 1.15 1.84 0.30 0.73 0.14 0.90 0.17 11.17 1.55 14.53 2.88 1.92 0.26c 37.29 5.33 3.97 0.53c 0.53 0.07 0.06 0.02 0.15 0.03 1.63 0.37 5.09 0.65c 3.03 0.66 0.03 0.01c 0.18 0.04 0.77 0.26c 0.45 0.11 0.42 0.08 0.44 0.10a 27.90 1.91 19.63 3.48 52.34 4.88 7.78 0.37c 44.54 5.14 5.75 0.86c
0.62 0.15a 0.55 0.08ab 13.50 1.24 2.19 0.26 0.60 0.07a 0.77 0.09a 11.71 2.69 14.29 2.37 1.02 0.12ab 43.34 5.22a 2.00 0.12ab 0.58 0.12 0.05 0.00 0.22 0.22 1.55 0.24 3.30 0.53ab 1.86 0.35a 0.02 0.00b 0.24 0.05 0.40 0.05b 0.52 0.11 0.34 0.08 0.32 0.07 28.15 2.65 18.59 2.73 53.06 5.01 4.26 0.44ab 48.77 5.45 11.65 2.64ab
0.038 0.005 0.956 0.133 0.048 0.007 0.371 0.776 0.002 0.029 0.001 0.692 0.194 0.539 0.241 0.003 0.007 0.025 0.084 0.020 0.05 0.103 0.019 0.467 0.589 0.543 ,0.001 0.075 ,0.001
whereas concentrations of serine in follicular fluid were negatively correlated with both insulin (0.50588) and glucose (0.73517) in circulation. Tryptophan was only correlated with circulating concentrations of insulin (0.67059) but no other systemic metabolites. The fatty acids myristic acid (0.52353) and tricosanoic acid (0.68824) in follicular fluid (Table 4) were significantly correlated with circulating concentrations of IGF1 in circulation, whereas glucose and palmitoleic acid (0.50301) were also negatively correlated. A strong negative correlation between myristic acid (in FF; 0.77059) and insulin (in circulation) was also observed (P , 0.05). IGF1 in circulation was moderately correlated with cis-10-heptadecanoic acid (0.55294), linolenic acid (0.60294) and arachidonic acid (0.51765) in follicular fluid, whereas insulin was correlated with linolenic acid, arachidonic acid and DHA. Glucose was positively correlated with elaidic acid, arachidonic acid, arachidic acid and DHA. BHB concentrations in circulation were only positively associated with concentrations of linoleic acid (0.62039) in follicular fluid but were negatively correlated
with a large number of fatty acids (cis-10-heptadecanoic acid, 0.6071; elaidic acid, 0.59823; arachidonic acid, 0.50665; EPA, 0.70163 and DHA, 0.65289). Discussion The severity of NEB associated with the tremendous demand for nutrients during lactation has been associated with reduced fertility in dairy cows. Greater NEFA concentrations in circulation reflect a greater mobilisation of adipose tissue to support milk production. Garverick et al. (2013) reported that serum NEFA concentrations were lower, and plasma glucose concentrations were greater, during the early postpartum period in cows that subsequently became pregnant. The cause was independent of interval to first ovulation, suggesting an effect of oocyte quality or the reproductive tract environment. The lower fertility observed in high-producing dairy cows is the result of the complex interaction between the systemic insults of parturition and lactation and the sensitivity of the steps required for successful establishment and maintenance of
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Table 3. Correlation analysis of circulating concentrations of IGF1, insulin, glucose, BHB and NEFA in serum and concentrations of amino acids in the follicular fluid of the pre-ovulatory dominant follicle of dairy heifers (n 5 6), non-lactating (n 5 5) and lactating (n ¼ 6) dairy cows (35–45 days postpartum) *Significant correlations are designated with an asterisk when P , 0.05. AAA, a-aminoadipic acid; ABA, a-aminobutyric acid Amino acid
AAA ABA Alanine Asparagine Aspartic acid Glutamic acid Glutamine Glycine Histidine Hydroxyproline Isoleucine Leucine Lysine Methionine Ornithine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
R values IGF1 (ng mL1)
Insulin (mIU mL1)
Glucose (mmol L1)
BHB (mmol L1)
NEFA (mmol L1)
0.68* 0.54* 0.21 0.25 0.23 0.33 0.29 0.41 0.49 0.41 0.06 0.39 0.48 0.40 0.65* 0.27 0.47 0.33 0.26 0.29 0.07 0.22
0.54* 0.69* 0.06 0.10 0.24 0.22 0.01 0.35 0.44 0.11 0.15 0.57* 0.20 0.31 0.50* 0.46 0.11 0.51* 0.01 0.67 0.17 0.33
0.46 0.63* 0.08 0.32 0.71* 0.49* 0.04 0.43 0.34 0.35 0.06 0.71* 0.47 0.44 0.42 0.59* 0.46 0.74* 0.01 0.20 0.23 0.40
0.61* 0.53* 0.20 0.37 0.11 0.05 0.05 0.47 0.19 0.04 0.03 0.74* 0.28 0.52* 0.62* 0.61* 0.48 0.45 0.05 0.37 0.24 0.56*
0.55* 0.11 0.26 0.23 0.18 0.19 0.19 0.13 0.46 0.07 0.14 0.01 0.20 0.23 0.41 0.22 0.41 0.10 0.02 0.21 0.00 0.10
Table 4. Correlation analysis of circulating concentrations of IGF1, insulin, glucose and BHB in serum and concentrations of fatty acids in the follicular fluid of the pre-ovulatory dominant follicle of dairy heifers (n 5 6), non-lactating (n 5 5) and lactating (n 5 6) dairy cows (35–45 days postpartum) *Significant correlations are designated with an asterisk when P , 0.05. EPA, cis-5, 8, 11, 14, 17-eicosapentaenoic acid; DHA, cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid Fatty acid
14 : 0 15 : 0 16 : 1 16 : 0 17 : 1 17 : 0 8 : 3n6 18 : 2n6 18 : 1n9c 18 : 3n3 18 : 1n9t 18 : 0 20 : 4n6 20 : 5n3 20 : 3n6 20 : 2 20 : 0 21 : 0 22 : 6n3 22 : 0 23 : 0 24 : 1 24 : 0
R values
Myristic Pentadecanoic Palmitoleic Palmitic cis-10-Heptadecanoic Heptadecanoic g-Linolenic Linoleic Oleic Linolenic Elaidic Stearic Arachidonic EPA cis-8,11,14-Eicosatrienoic cis-11,14-Eicosadienoic Arachidic Heneicosanoic DHA Behenic Tricosanoic Nervonic Lignoceric
IGF1 (ng mL1)
Insulin (mIU mL1)
Glucose (mmol L1)
BHB (mmol L1)
0.52* 0.45 0.31 0.25 0.55* 0.49* 0 0.3 0 0.60* 0.47 0.31 0.52* 0.49* 0.46 0.11 0.21 0.03 0.34 0.19 0.69* 0.14 0.24
0.77* 0.41 0.27 0.13 0.36 0.36 0.06 0.32 0.16 0.67* 0.43 0.06 0.63* 0.38 0.31 0.14 0.18 0.44 0.54* 0.38 0.38 0.16 0.26
0.43 0.34 0.50* 0.02 0.36 0.24 0.04 0.23 0.18 0.4 0.49* 0.06 0.55* 0.44 0.12 0.12 0.63* 0.3 0.53* 0.08 0.19 0.25 0.33
0.41 0.19 0.09 0.43 0.61* 0.39 0.16 0.62* 0.49 0.49 0.60* 0.09 0.51* 0.70* 0.05 0.05 0.02 0.27 0.65* 0.25 0.33 0.22 0.06
Follicular-fluid metabolome in dairy cattle
pregnancy to these insults. This study aimed to determine how the metabolic stress of lactation affects the microenvironment to which the pre-ovulatory oocyte is exposed by examining the amino-acid and fatty-acid profiles of follicular fluid from the pre-ovulatory follicle. Analysis of circulating metabolites of non-lactating and lactating postpartum dairy cows revealed that lactation was associated with significantly decreased insulin, IGF1 and glucose concentrations and increased NEFA and BHB concentrations. Non-lactating cows had higher insulin, glucose and IGF1 and lower NEFA and BHB in circulation from 45 to 65 dpp compared with non-lactating dairy cows, similar to what was observed in maiden heifers. The metabolic status of the animal was reflected in the concentrations of certain amino acids and fatty acids in the follicular fluid of the pre-ovulatory follicle. The bodyweight and BCS of non-lactating and lactating cows were significantly lower than nulliparous heifers throughout the postpartum period. The overall profiles for these two parameters were similar between lactating and non-lactating cows; however, a significant day*treatment interaction was observed for both parameters, caused by an inversion of profiles between Day 28 and Day 35 postpartum. The reason for this inversion is unclear. However, it should be noted that up to the time of follicular-fluid aspiration at approximately Day 35, the profiles were not different. Both the fatty-acid and amino-acid composition of FF were similar between the non-lactating cows and heifers, reflecting the similarity in their metabolic profiles at the time of follicularfluid aspiration. In contrast, in lactating cows, the impact of lactation was reflected in the follicular-fluid composition, which was significantly different with respect to the concentration of specific amino and fatty acids from that of non-lactating cows and maiden heifers. Previous studies have demonstrated good correlation between circulating and follicular-fluid concentrations of metabolites such as glucose, BHB, NEFA and triglyceride (Leroy et al. 2004a). Indeed, the hypothesis proposed by Britt (1992) states that growth of the oocyte in the follicle during the period of negative energy balance has a detrimental effect on oocyte competence. While dietary composition does significantly affect the metabolic status of an animal, available evidence indicates that the consequence of metabolic stress, as evidenced by changes in circulating metabolites, is reduced fertility, which is supported by the present data i.e. the impact of gestation and parturition in the non-lactating cows and any effect it had on the follicular-fluid microenvironment was no longer evident as early as 35–45 dpp, when both the circulating metabolite concentrations and the follicular-fluid composition were similar to those of a maiden heifer. The most abundant amino acids in the follicular fluid of the pre-ovulatory dominant follicle were glycine, alanine and glutamine. The high abundance of alanine is consistent with previous studies in which the dominant follicle of the first follicular wave as well as the pre-ovulatory dominant follicle in heifers had higher concentrations of alanine compared with subordinate follicles of either the first or second follicular wave (Sarty et al. 2006). Moreover, alanine was detected in the pre-ovulatory follicular fluid of all heifers and non-lactating cows i.e. presumed good fertility, whereas it was only detected in the follicular fluid of one of six lactating cows i.e. the less fertile group.
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In a previous study, glycine concentrations were higher in the follicular fluid of follicles whose oocytes developed to the blastocyst stage after in vitro fertilisation (Matoba et al. 2014). This may seem contradictory to the results of this study as glycine concentrations were higher in follicular fluid from lactating cows (compromised fertility) compared with the nonlactating cows and heifer group; however, the data generated in the study by Matoba et al. (2014) were from follicles dissected from abattoir-derived ovaries i.e. small follicles at undefined stages of the follicle wave. Samples from this study were generated from the pre-ovulatory follicle and data indicate that there are significant differences in the metabolic component of follicular fluid from follicles at different stages of the follicle wave (Sarty et al. 2006). Analysis of culture media recovered from bovine oocytes matured in vitro revealed significant differences in the depletion and appearance of amino acids between oocytes that failed to cleave and those that developed to the 16-cell stage (Hemmings et al. 2012). Oocytes that failed to cleave depleted increased amounts of glutamine, arginine and leucine. Consistent with this, leucine concentrations were significantly lower in the follicular fluid of lactating cows compared with the other two groups. In addition to lower concentrations of leucine in the follicular fluid of postpartum lactating dairy cows, valine, methionine, phenylalanine, ornithine, lysine, histidine and tyrosine were all lower in the follicular fluid of lactating dairy cows. Correlation analysis also revealed that higher concentrations of BHB in circulation, as is the case in the lactating group, were negatively associated with leucine, valine, methionine, phenylalanine and ornithine amino-acid concentrations in follicular fluid. Moreover concentrations of AAA, ABA, serine and proline were all higher in lactating cows, with a positive correlation between concentrations of AAA and ABA in follicular fluid and BHB concentrations in circulation. While these results should be interpreted with caution due to the low numbers of animals involved, the data suggest that a follicular-fluid profile similar to that observed in the lactating dairy cow is not as supportive an environment for successful oocyte competency. The fatty acids linoleic, oleic, stearic and palmitic acids were the most abundant fatty acids detected in the follicular fluid of all three groups. This is similar to what was observed in the follicular fluid of dairy heifers and lactating cows ,80 dpp (Bender et al. 2010). Exposure to palmitic or stearic acid during in vitro maturation of oocytes decreased developmental competence after fertilisation but oleic acid exposure could compensate for this decrease (Aardema et al. 2011). None of these were significantly different in our model but have been previously shown to be higher in the follicular fluid of dairy cows compared with maiden heifers (Bender et al. 2010). The discrepancies between these data and this study may be explained, in part, by the nature of the models used. In the study by Bender et al. (2010) the lactating dairy cows were in an average lactation of 3.75, were ,80 dpp at the time of follicular-fluid collection and the fatty-acid data presented was from dominant follicles at three different stages of development. Linoleic acid, as well as the less-abundant linolenic acid, was affected by the metabolic status of the animal in this study. In feeding supplementation studies, the addition of linoleic acid
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had a negative effect on fertility compared with supplementation with linolenic acid (reviewed by Santos et al. 2008). Supplementation of oocyte maturation media in vitro with linolenic acid increased both maturation of oocytes and blastocyst formation (Marei et al. 2009) whereas linoleic-acid supplementation reduced nuclear maturation, cumulus expansion and subsequent embryo development (Marei et al. 2010). The lower concentration of linolenic acid coupled with higher concentrations of linoleic acid in the follicular fluid of lactating cows compared with both the non-lactating cows and heifers may contribute to reduced fertility observed in the postpartum dairy cow, probably mediated though their effects on the pre-ovulatory oocyte. Of the other abundant fatty acids detected in the follicular fluid, previous studies have shown that addition of stearic acid to culture media results in decreased cleavage rates and blastocyst development while fertilisation rates were lower in oocytes exposed to palmitic or stearic acid (Leroy et al. 2005). We observed no difference in the concentrations of stearic, oleic or palmitic acid in the follicular fluid of lactating cows compared with either non-lactating cows or maiden heifers. These results may seem at odds; however, it is important to note that in the study of Leroy et al. (2005) follicular-fluid NEFA concentrations were compared between Days 16 and 44 postpartum. This is consistent with our data, where NEFA concentrations in circulation are hugely different on Day 16 postpartum. However, by Days 35–45 postpartum, when the follicular-fluid samples were collected in the present study, NEFA concentrations in lactating cows were coming back to basal levels (Fig. 1f ). Concentrations of myristic, pentadecanoic, elaidic and arachidonic acids and the total concentration of n3 PUFAs were higher while cis-10-heptadecanoic and heptadecanoic acids were lower in lactating compared with non-lactating cows, maiden heifers or both. This would support the notion that different compositions of fatty acids in the follicular fluid contribute to reduced fertility in postpartum dairy cows. In conclusion, lactation induces distinct changes in the overall metabolic status of postpartum dairy cows compared with non-lactating cows and maiden heifers These changes are associated with divergent metabolite profiles in the follicular fluid; for example, lactation induced increases in the abundance of the amino acids AAA, ABA, glycine, serine and proline, as well as the fatty acids myristic acid, linoleic acid and the ratio of n6 : n3 PUFAs, and a decrease in leucine, methionine, phenylalanine, ornithine, lysine, histidine, tyrosine, valine and in pentadecanoic, cis-10-heptadecanoic, heptadecanoic, linolenic, elaidic, arachidonic, heneicosanoic acids plus EPA, DHA and the total concentration of n3 PUFAs. These changes create a different microenvironment for the developing oocyte and presumably contribute to lower fertility in some cases. It is possible that addition of some of these factors (within physiological ranges) to in vitro maturation media, may help increase blastocyst rates both in cattle as well as in other species. Acknowledgements We wish to acknowledge the help of staff and students who assisted in sample collection. The research leading to these results has received funding from the European Union Seventh Framework Program FP7/2007–2013 under grant agreement n8 312097 (‘FECUND’).
N. Forde et al.
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