The use of ensiled olive cake in the diets of Friesian cows increases beneficial fatty acids in milk and Halloumi cheese and alters the expression of SREBF1 in adipose tissue.

This study aimed to evaluate the effect of dietary inclusion of ensiled olive cake, a by-product of olive oil production, on milk yield and composition and on fatty acid (FA) profile of milk and Halloumi cheese from cows. Furthermore, the effect of olive cake on the expression of selected genes involved in mammary and adipose lipid metabolism was assessed in a subset of animals. A total of 24 dairy cows in mid lactation were allocated into 2 isonitrogenous and isoenergetic feeding treatments, named the control (CON) diet and the olive cake (OC) diet, in which part of the forages (alfalfa, barley hay, and barley straw) were replaced with ensiled OC as 10% of dry matter according to a 2 × 2 crossover design with two 28-d experimental periods. At the end of the second experimental period, mammary and perirenal adipose tissue samples were collected from 3 animals per group for gene expression analysis by quantitative reverse-transcription PCR. The expression of 11 genes, involved in FA synthesis (ACACA, FASN, G6PDH), FA uptake or translocation (VLDLR, LPL, SLC2A1, CD36, FABP3), FA saturation (SCD1), and transcriptional regulation (SREBF1, PPARG), was evaluated. No significant differences were observed between groups concerning milk yield, fat percentage, protein percentage, and protein yield (kg/d), whereas milk fat yield (kg/d) increased in the OC group. Dietary supplementation with ensiled OC modified the FA profile of milk and Halloumi cheese produced. There was a significant decrease in the concentration of de novo synthesized FA, saturated FA, and the atherogenic index, whereas long-chain and monounsaturated FA concentration was increased in both milk and cheese. Among individual saturated FA, only stearic acid was elevated, whereas among individual monounsaturated FA, increments of oleic acid (C18:1 cis-9) and the sum of C18:1 trans-10 and trans-11 acids were demonstrated in milk and Halloumi cheese produced. Although no diet effect was reported on total polyunsaturated FA, the concentration of CLA cis-9,trans-11 was increased in both milk and Halloumi cheese fat of the OC group. The expression of the genes tested was unaffected apart from an observed upregulation of SREBF1 mRNA expression in perirenal fat from cows fed the OC diet. Milk FA differences observed were not associated with alterations in mammary expression of genes involved in FA synthesis, uptake, translocation, and regulation of lipogenesis. Overall, the inclusion of ensiled OC in cow diets for a 4-wk period improved, beneficially for human health, the lipid profile of bovine milk and Halloumi cheese produced without adversely affecting milk yield and composition or the expression of genes involved in lipid metabolism of mammary and adipose tissues in cows.

Variants in the 3' untranslated region of the ovine acetyl-coenzyme A acyltransferase 2 gene are associated with dairy traits and exhibit differential allelic expression.

The acetyl-CoA acyltransferase 2 (ACAA2) gene encodes an enzyme of the thiolase family that is involved in mitochondrial fatty acid elongation and degradation by catalyzing the last step of the respective ß-oxidation pathway. The increased energy needs for gluconeogenesis and triglyceride synthesis during lactation are met primarily by increased fatty acid oxidation. Therefore, the ACAA2 enzyme plays an important role in the supply of energy and carbon substrates for lactation and may thus affect milk production traits. This study investigated the association of the ACAA2 gene with important sheep traits and the putative functional involvement of this gene in dairy traits. A single nucleotide substitution, a T to C transition located in the 3' untranslated region of the ACAA2 gene, was used in mixed model association analysis with milk yield, milk protein yield and percentage, milk fat yield and percentage, and litter size at birth. The single nucleotide polymorphism was significantly associated with total lactation production and milk protein percentage, with respective additive effects of 6.81 ± 2.95 kg and -0.05 ± 0.02%. Additionally, a significant dominance effect of 0.46 ± 0.21 kg was detected for milk fat yield. Homozygous TT and heterozygous CT animals exhibited higher milk yield compared with homozygous CC animals, whereas the latter exhibited increased milk protein percentage. Expression analysis from age-, lactation-, and parity-matched female sheep showed that mRNA expression of the ACAA2 gene from TT animals was 2.8- and 11.8-fold higher in liver and mammary gland, respectively. In addition, by developing an allelic expression imbalance assay, it was estimated that the T allele was expressed at an average of 18% more compared with the C allele in the udder of randomly selected ewes. We demonstrated for the first time that the variants in the 3' untranslated region of the ovine ACAA2 gene are differentially expressed in homozygous ewes of each allele and exhibit allelic expression imbalance within heterozygotes in a tissue-specific manner, supporting the existence of cis-regulatory DNA variation in the ovine ACAA2 gene. This is the first study reporting differential allelic imbalance expression of a candidate gene associated with milk production traits in dairy sheep.

Partial suckling of lambs reduced the linoleic and conjugated linoleic acid contents of marketable milk in Chios ewes.

The objective of this work was to investigate the effect of weaning systems applied in a commercial dairy sheep farm on the fatty acid (FA) composition of marketable milk produced. Forty second parity, purebred Chios ewes were allocated to the following weaning treatments: (a) ewes were weaned from their lambs at 48 h after birth and machine milked twice daily [no lambs (NL) group, n=20]; or, (b) starting 48 h postpartum, ewes were separated from their lambs for 12h during the evening, machine milked once daily the following morning, and lambs were allowed to suckle for 12 h during the day for the first 5 wk of lactation [partial suckling (PS) group, n=20]. After weaning of the PS lambs at wk 6 of age, all ewes were machine milked twice daily. Commercial milk yield and milk composition was recorded weekly (fat, protein, FA content) or fortnightly (somatic cell counts) throughout the first 10 wk of lactation. The PS ewes compared with NL group produced commercial milk lower in milk yield, milk fat, and somatic cell counts, but not in protein content during the first 5-wk period. Such differences were not observed after weaning of the PS lambs. The FA profile of commercial milk was also affected by partial suckling during the preweaning period. Total polyunsaturated FA were higher in NL compared with PS ewe milk at wk 1, 2, 4, and 5 (on average, 21% higher), whereas no differences were detected between NL and PS ewe milk from wk 6 to 10 of lactation. From the polyunsaturated FA, linoleic acid (C18:2 cis-9,cis-12) and conjugated linoleic acid (C18:2 cis-9,trans-11; rumenic acid) were particularly affected, showing on average a reduction of 18 and 38%, respectively. From the monounsaturated FA, vaccenic acid (C18:1 trans-11) was affected during wk 1 and 2 of the treatment period, with the PS ewe milk having reduced content compared with the NL milk. Other unsaturated FA, such as oleic acid and α-linolenic acid, or saturated FA were not found to be affected by the weaning treatment. The results indicate that partial suckling of lambs during the first 5 wk of lactation adversely affected both the total fat content of milk obtained by machine milking of their dams and the unsaturation content of the milk fat. Particularly affected were the linoleic acid and cis-9,trans-11 conjugated linoleic acid content of ewe milk.

Assessment of inbreeding resulting from selection for scrapie resistance: a model for rare sheep breeds.

Inbreeding in a small population of Chios sheep undergoing intense selection for the PrP gene was assessed 10 years after the beginning of a scrapie resistance selection programme. Inbreeding in this stock, already under selection for production traits, was analysed by using pedigree records containing 10,492 animals from 1968 to 2008, and also by genotyping 192 individuals with a panel of 15 microsatellites. Genetic markers indicated a loss of heterozygosity (FIS over all loci was 0.059) and allelic diversity (mean effective number of alleles was 3.075±0.275). The annual rate of inbreeding increased significantly after the start of the scrapie resistance programme, ΔF=0.005 compared with ΔF=0.001 before 1999, and was subjected to several genetic bottlenecks, mainly due to the low initial frequency of resistant animals. However, the mean individual inbreeding coefficient estimated from the pedigree - in this closed stock resembling the case of a rare breed - stood at the level of 4.5 per cent, five generations after the implementation of selection for the PrP gene. The inbreeding coefficient estimated by genetic markers was 4.37 per cent, implying that such a marker panel could be a useful and cost-effective tool for estimating inbreeding in unrecorded populations.

A single nucleotide polymorphism in the acetyl-coenzyme A acyltransferase 2 (ACAA2) gene is associated with milk yield in Chios sheep.

The objective of this work was to identify single nucleotide polymorphisms (SNP) in the ovine acetyl-coenzyme A acyltransferase 2 (ACAA2) gene and investigate their association with milk production traits. Molecular characterization was accomplished by sequencing the entire coding region and the 3' and 5' untranslated regions (UTR) of the ACAA2 gene from individuals of the Chios sheep bred in Cyprus. All exons appeared to be monomorphic except for the last exon, for which a single SNP located in the 3' UTR of the gene (HM537015:g.2982T>C) was detected. Mixed model association analysis, using SNP data from 318 animals from 104 paternal half-sib families and first-lactation phenotype and pedigree information on 2,405 ewes revealed that this SNP was significantly associated with milk yield. The significance of the SNP effect persisted when milk yield information up to the third lactation was analyzed. Both alleles at the locus segregated at similar frequencies. The T allele was associated with increased milk yield and exhibited partial dominant action. Animals with the g.2982TT or g.2982CT genotype had significantly higher milk yield than those with the g.2982CC genotype, with the g.2982T allele having an additive effect of 13.4 (± 4.7) kg and a dominance effect of 7.9 (± 6.1) kg. Based on estimated allelic effects and sample allele frequencies, the g.2982T>C SNP explained 10% of the additive genetic variance for milk yield. A putative mode of action through nutrient metabolism is discussed.

Technical note: A simplified PCR-based assay for the characterization of two prolactin variants that affect milk traits in sheep breeds.

In the present study, a rapid and cost-effective PCR-based assay was developed for the genetic identification of 2 different variants within intron 2 of the prolactin gene. This polymorphism has previously been associated with milk traits in some ovine breeds and was recently proposed as a potential marker for future breeding schemes in dairy sheep. Until now, 2 alleles (A and B) have been identified by PCR-RFLP that included HaeIII digestion of a 2.5-kb PCR fragment. By partial sequencing of the prolactin gene intron 2, it was found that the B variant results from a 23-bp deletion of the A variant of the prolactin gene and not from an extra HaeIII digestion site, as had been reported. This finding assisted the design of new primers for analysis of prolactin intron 2 variants based on the size of an easily amplified short PCR product, thereby avoiding the need and cost for additional digestions. The method was validated by genotyping 80 animals from 2 breeds and showed 100% sensitivity and specificity compared with the PCR-RFLP assay. The established simplified PCR assay was then successfully used to genotype 356 Chios sheep.

The use of chicory for parasite control in organic ewes and their lambs.

The aim of the present study was to investigate the potential benefits of grazing lactating ewes and their lambs on chicory (Cichorium intybus). Fifty-six certified organic twin-rearing ewes were either drenched with an anthelmintic or not, within 2 days after parturition and were grazed upon either grass/clover or chicory pastures. Around 12 weeks after parturition a subset of 12 lambs per treatment was slaughtered for worm number and parasite species determination. The faecal egg counts of lambs from undrenched ewes grazing on chicory were significantly lower than those of lambs from undrenched ewes grazing on grass. Lambs grazing on chicory had similar abomasal worm counts as those grazing on grass at 12 weeks of age; the predominant species was Teladorsagia circumcincta. There was no difference between the intestinal worm counts in lambs grazing on grass or chicory, with Trichostrongylus vitrinus being the predominant species. Liveweight gains over the 126-day experimental period were significantly higher in lambs from drenched than those from undrenched ewes. Lambs from undrenched ewes grazing on chicory had higher liveweight gains compared to those from undrenched ewes grazing on grass. Although chicory grazing did not affect ewe nematode egg excretion, it resulted in lower egg counts in lambs and improved their liveweight gains to the same level as those deriving from drenched ewes.

The effect of chicory ( Cichorium intybus ) and sulla ( Hedysarum coronarium ) on larval development and mucosal cell responses of growing lambs challenged with Teladorsagia circumcincta.

The aim of this study was to investigate the effects of grazing different bioactive forages on acquired immunity against Teladorsagia circumcinta infection. The development of immunity was assessed by following the response of trickle-infected lambs grazing chicory (Cichorium intybus; IC), sulla (Hedysarum coronarium; IS) or grass/clover (Lolium perenne/Trifolium repens; IGC), to a single challenge infection. Parasite-naive lambs, grazing grass/clover, were also challenged with the single infection dose providing the uninfected control (UGC) group. Trickle infection significantly reduced worm establishment, inhibited larval development and increased mucosal mast cell (MMC) and globule leucocyte (GL) cells. Grazing treatment (chicory, sulla or grass/clover) significantly affected adult worm (P<0.05), late-L4 (P<0.01) and mid-L4 (P<0.01) larval-stage recoveries of the trickle-infected lambs, with IGC group always carrying higher worm burdens than either IC or IS lambs. MMC and GL cells of trickle-infected lambs were positively correlated with the proportion of early-L4 worms recovered and negatively correlated with both the proportion of adult worms recovered and the total worm establishment, suggesting that the observed effects were due to an enhanced immune response. The results suggest elevated immune responses against T. circumcincta infections in growing lambs grazing on either sulla or chicory compared to those grazing on grass/clover, probably due to differences in forage nutritional values.

Testing for direct anthelmintic effects of bioactive forages against Trichostrongylus colubriformis in grazing sheep.

The aim of the present study was to investigate potential direct anthelmintic effects of forages that contain plant secondary metabolites (PSM) towards the intestinal nematode Trichostrongylus colubriformis. For this purpose, we introduced an experimental design, which enabled us to investigate the direct anthelmintic effects of PSM-rich forages, without significant interference of possible indirect immunological effects of PSM. Sixty parasite naive sheep were infected with 8000 T. colubriformis L(3) on day 1 of the experiment. On day 28, sheep entered one of the experimental plots, which consisted of four PSM-rich forages and one control: Lotus pedunculatus (lotus), Hedysarium coronarium (sulla), Onobrychis viciifolia (sainfoin), Cichorium intybus (chicory) and Lolium perenne/Trifolium repens (grass/clover). On day 35 of the experiment, all sheep were re-infected with 8000 T. colubriformis L(3) and were killed on day 42. By day 42, all parasites of the primary infection would have been recovered as adults, whereas those of the secondary challenge would have only developed to the fourth stage larvae within a week (i.e. days 35-42). The first batch of larvae would enable us to investigate possible direct anthelmintic effects of PSM-rich forages against adult T. colubriformis, whereas the second one whether grazing on the PSM-rich forages could affect the establishment of the incoming infective larvae. Sheep grazing on lotus tended to have a lower FEC compared to sheep grazing on grass/clover (P = 0.06), whereas daily faecal output was higher in sheep grazing lotus compared to those grazing on the other forages (P < 0.05). As a consequence, daily egg output was similar in all sheep. Grazing on the PSM-rich forages for a period of 2 weeks did not affect the immature and adult parasite populations. Although the present experimental design enabled us to test the experimental hypothesis, the lack of evidence on a direct anthelmintic effect of PSM-rich forages can not be considered conclusive, as the composition of PSM-rich forages is variable within and across grazing seasons.

The consequences of short-term grazing of bioactive forages on established adult and incoming larvae populations of Teladorsagia circumcincta in lambs.

The objective of this study was to investigate the consequences of short-term grazing on bioactive forages on (i) the viability and fecundity of established adult Teladorsagia circumcincta population and (ii) the establishment and development of incoming T. circumcincta infective larvae. Forty-eight, parasite naive, 3-month old, grazing lambs were artificially infected with 8000 infective larvae of T. circumcincta on day 1 of the experiment. On day 21 p.i., lambs were allocated to one of three bioactive forage grazing treatments; chicory (Cichorium intybus), sulla (Hedysarum coronarium), lotus (Lotus pedunculatus), and the control grass/clover (Lolium perenne/Trifolium repens) forage. On day 28 of the experiment a second dose of 8000 T. circumcincta infective larvae was administered to the lambs to investigate the effects of forages on the ability of infective larvae to establish within the host. All animals were slaughtered for worm recovery on day 35, while liveweight gain, feacal egg counts (FEC) and total worm egg output were monitored regularly throughout the experiment. Although FEC or total egg output were similar among the groups, adult worm burdens at slaughter were significantly affected (P<0.05) by forage treatment during the 2 week grazing period. Lambs grazing chicory had the lowest adult worm burdens and significantly lower numbers of male worms compared to those grazing on grass/clover (P<0.01), while the lambs grazing on sulla or lotus had similar adult populations to grass/clover fed animals. The results from the worm recoveries of the second dose (immature worm burdens) were affected by physiologically and/or immunologically mediated mechanisms, which reduced larval establishment in all treatments. Nevertheless, immature worm burdens at slaughter were similar between chicory, sulla and grass/clover group, while the immature worm recoveries from the lotus group were significantly higher (P<0.05) compared to those from lambs grazing grass/clover. Overall, the results of the present study support the view that chicory can be a promising candidate species in pasture management practices to control T. circumcincta burdens.