The forgotten variable? Does the euthanasia method and sample storage condition influence an organisms transcriptome - a gene expression analysis on multiple tissues in pigs.

BACKGROUND: Transcriptomic studies often require collection of fresh tissues post euthanasia. The chosen euthanasia method might have the potential to induce variations in gene expressions that are unlinked with the experimental design. The present study compared the suitability of 'nitrogen gas in foam' (ANOXIA) in comparison to a non-barbiturate anaesthetic, T-61® (T61), for euthanizing piglets used in transcriptome research. Further, the effect of common tissue storage conditions, RNAlater™ (RL) and snap freezing in liquid nitrogen (LN2), on gene expression profiles were also analysed. RESULTS: On comparison of the 3'mRNA-Seq data generated from pituitary, hypothalamus, liver and lung tissues, no significant differential expression in the protein coding genes were detected between the euthanasia methods. This implies that the nitrogen anoxia method could be a suitable alternative for euthanasia of piglets used in transcriptomic research. However, small nuclear RNAs (snRNAs) that constitute the eukaryotic spliceosomal machinery were found to be significantly higher (log2fold change ≥ 2.0, and adjusted p value ≤ 0.1) in pituitary samples collected using ANOXIA. Non-protein coding genes like snRNAs that play an important role in pre-mRNA splicing can subsequently modify gene expression. Storage in RL was found to be superior in preserving RNA compared to LN2 storage, as evidenced by the significantly higher RIN values in representative samples. However, storage in RL as opposed to LN2, also influenced differential gene expression in multiple tissues, perhaps as a result of its inability to inhibit biological activity during storage. Hence such external sources of variations should be carefully considered before arriving at research conclusions. CONCLUSIONS: Source of biological variations like euthanasia method and storage condition can confound research findings. Even if we are unable to prevent the effect of these external factors, it will be useful to identify the impact of these variables on the parameter under observation and thereby prevent misinterpretation of our results.

Effect of temperature manipulation during incubation on body weight, plasma parameters, muscle histology, and expression of myogenic genes in breast muscle of embryos and broiler chickens from two commercial strains.

1. This study evaluated the effect of a higher incubation temperature on body weight, plasma profile, histology and expression of myogenin (MYOG), insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor A (VEGFA) genes in breast muscle of embryos and broilers from two commercial strains.2. A total of 784 eggs from Ross 308 and Cobb 500 broiler breeder flocks were used. Half of the eggs per strain were incubated at control temperature (37.8°C), whereas the other half were exposed to heat treatment (HT) of 38.8°C between embryonic day (ED) 10 and 14, for 6 h/day. Embryos and chicks were sampled on ED 19 and at hatch. A total of 480, one-day-old chicks per strain and incubation temperature were reared up to 42 d post-hatch.3. The HT increased hatch weight of Ross chicks and 42-d body weight of broilers from both strains. Lower plasma triacylglycerol levels were measured for HT embryos and broilers on ED 19 and 42 d post-hatch, respectively. HT reduced plasma T3 levels in Ross embryos and broilers for the same periods. Hepatic TBARS concentrations were elevated by HT compared to the control incubation.4. The HT reduced breast muscle VEGFA gene expression of Cobb embryos on ED 19, whereas expression was stimulated in day-old chicks. At 42 d post-hatch, fibre area was increased by HT regardless of strain. Compared to the control incubation, HT increased the breast yield of Ross broilers and leg yield of Cobb. Ross-HT broilers had a higher pH at 24 h after slaughter and better water holding capacity than Cobb-HT broilers.5. These results suggested that HT increased body weight, fibre area, IGF-I gene expression and lowered plasma triacylglycerol levels of broiler chickens from both strains at 42 d. However, HT influenced the expression of VEGF-A and MYOG genes and meat quality differently between the broiler strains.

Sequencing refractory regions in bird genomes are hotspots for accelerated protein evolution.

BACKGROUND: Approximately 1000 protein encoding genes common for vertebrates are still unannotated in avian genomes. Are these genes evolutionary lost or are they not yet found for technical reasons? Using genome landscapes as a tool to visualize large-scale regional effects of genome evolution, we reexamined this question. RESULTS: On basis of gene annotation in non-avian vertebrate genomes, we established a list of 15,135 common vertebrate genes. Of these, 1026 were not found in any of eight examined bird genomes. Visualizing regional genome effects by our sliding window approach showed that the majority of these "missing" genes can be clustered to 14 regions of the human reference genome. In these clusters, an additional 1517 genes (often gene fragments) were underrepresented in bird genomes. The clusters of "missing" genes coincided with regions of very high GC content, particularly in avian genomes, making them "hidden" because of incomplete sequencing. Moreover, proteins encoded by genes in these sequencing refractory regions showed signs of accelerated protein evolution. As a proof of principle for this idea we experimentally characterized the mRNA and protein products of four "hidden" bird genes that are crucial for energy homeostasis in skeletal muscle: ALDOA, ENO3, PYGM and SLC2A4. CONCLUSIONS: A least part of the "missing" genes in bird genomes can be attributed to an artifact caused by the difficulty to sequence regions with extreme GC% ("hidden" genes). Biologically, these "hidden" genes are of interest as they encode proteins that evolve more rapidly than the genome wide average. Finally we show that four of these "hidden" genes encode key proteins for energy metabolism in flight muscle.

The influence of thyroid state on hypothalamic AMP-activated protein kinase pathways in broilers.

To investigate whether there are important interactions in play in broilers between thyroid hormones and the central regulation of energy homeostasis through AMP-activated protein kinase (AMPK), we induced a functional hyperthyroid and hypothyroid state in broiler chicks, and quantified systemic and hypothalamic AMPK related gene expression and related protein. Thyroid state was manipulated through dietary supplementation of triiodothyronine (T3) or methimazole (MMI) for 7 days. A hypothalamic AMPK suppressor, 0.1% α-lipoic acid (α-LA) was used to assess the effects of the T3 and MMI feed formulations on the AMPK pathways. Feed intake and body weight were reduced in both hypothyroid and hyperthyroid conditions. In hyperthyroid conditions (T3 supplementation) expression of the AMPKα1 subunit increased, while in hypothyroid conditions (MMI supplementation) active phosphorylated AMPK levels in the hypothalamus dropped, but gene expression of the AMPKα1 and α2 subunit increased. For FAS and ACC (involved in fatty acid metabolism), and CRH, TRH and CNR1 (anorexigenic neuropeptides stimulating energy expenditure) there were indications that their regulation in response to thyroid state might be modulated through AMPK pathways. Our results indicate that the expression of hypothalamic AMPK as well as that of several other genes from AMPK pathways are involved in thyroid-hormone-induced changes in appetite, albeit differently according to thyroid state.

Direct and maternal reduced balanced protein diet influences the liver transcriptome in chickens.

The objective of this study was to evaluate, by means of RNA sequencing, the direct and transgenerational effect of a reduced balanced protein (RP) diet on broiler breeder metabolism. Chickens of the F0 generation were fed a control (C) or RP diet, and their F1 progeny was fed a C or RP diet as well, resulting in four groups of chickens: C/C, C/RP, RP/C and RP/RP. While both direct and maternal effects were seen on body weight, breast muscle weight and abdominal fat weight in the F1 generation, the direct effect was the most dominant one. The liver transcriptome in the F1 generation showed that amino acid metabolism was up-regulated in chickens that received the control feed when compared with their respective contemporaries that received the reduced protein diet. Interestingly, chickens hatched from control-fed hens but reared on the reduced protein diet (C/RP group) activated a fatty acid metabolism, expressing more fatty acid desaturase 1 gene, fatty acid desaturase 2 gene and elongation of very long-chain fatty acids protein 2 gene, when compared with control-fed chickens hatched from control-fed hens (C/C group), while chickens hatched from reduced protein-fed hens that received themselves the same reduced protein diet (RP/RP group) triggered their glucose metabolism more, showing elevated levels of phosphofructokinase gene, 6-phosphofructo-2-kinase/fructose-2,6-biphospatase 4 and fructose-biphosphate aldolase C mRNA compared with the chickens hatched from reduced protein-fed hens but reared on a control diet (RP/C group). This suggests that the maternal protein diet has an impact on the metabolism of broilers when they are reared on a RP diet.

Management factors resulting in a severe reduction in feed intake-induced spiking mortality syndrome in young broiler chicks.

This study aimed to induce spiking mortality syndrome (SMS) in 10-day-old broiler chicks by changing feed particle size (crumble feed to pellet feed) and/or feed source location (from a small feeder at the pen's center to a large feeder at the front of the pen), followed by full day feed deprivation of all broiler chicks on day 11. In total, 396-day-old male Ross 308 broiler chicks were randomly assigned to 4 treatments (Con: without change in feed particle size and feed source location; Par: changing crumble feed to pellet feed on day 10; Loc: changing feed source location on day 10; LocPar: changing both feed particle size and feed source location on day 10). Each treatment consisted of 9 replicate pens with 11 chicks each. Each treatment was applied at 09:00 on days 10 and 11. On both days, chicks with SMS were identified based on clinical symptoms (down in sternal or lateral recumbency, hyperventilation). Plasma glucose, 3, 3', 5-triiodothyronine (T3), thyroxine (T4) concentrations, insulin, and liver glycogen concentrations of chicks without (normal) and with SMS were measured. Proportional organ and digestive tract including content weights were recorded. Broiler behavior was assessed hourly from 08:30 to 17:30 on day 10. On day 10, the Par, Loc, and LocPar groups spent significantly less time feeding and more time lying down compared with the Con group. On days 10 and 11, SMS clinical signs were observed around 2.5 to 3.5 h after the initiation of treatments, and the Loc group had the most SMS morbidity level. Spiking mortality syndrome chicks had significantly less digestive tract contents compared with Normal chicks on day 10. Spiking mortality syndrome was induced successfully with the treatments, according to their significantly reduced plasma glucose, insulin, T3 and T4 concentrations as well as liver glycogen content. A significant correlation between plasma glucose and liver glycogen was observed in SMS chicks. In conclusion, management factors inducing the reduction or absence of feed intake on day 10 or day 11 can trigger the occurrence of SMS in young broiler chicks.

Arabinoxylan-oligosaccharides kick-start arabinoxylan digestion in the aging broiler.

While arabinoxylans (AX), an important dietary fiber fraction of wheat-based broiler diets, are known for exerting antinutritional effects in the gastrointestinal (GI) tract of broilers, the prebiotic potential of arabinoxylan-oligosaccharides (AXOS) is also well-documented. However, inconsistent performance responses as well as the effectiveness of low amounts of AXOS used in diets of previously conducted experiments put into question the classical prebiotic route being the sole mode of action of AXOS. The objective of this study was to investigate the effects of dietary AXOS addition on the rate of AX digestion in the gastrointestinal tract of broilers as a function of broiler age to gain more insight into the mode of action of these oligosaccharides. A feeding trial was performed on 480 one-day-old chicks (Ross 308) receiving a wheat-based diet supplemented with or without 0.50% AXOS, containing no endoxylanases. Digesta samples from ileum and caeca and fecal samples were analyzed for AX content, AX digestibility, intestinal viscosity, and microbial AX-degrading enzyme activities at 6 different ages (day 5, 10, 15, 21, 28, 35). Chicks fed from hatching with 0.50% AXOS demonstrated a higher ileal viscosity (P < 0.05). Also higher levels of AX solubilization and fermentation compared to control birds at 10 D were observed. This was noted by the higher total tract AX digestibility of water-extractable AX (WE-AX) and total AX (TOT-AX) at this age (P < 0.05). Although no significant difference in AX-degrading enzyme activities was observed among the dietary treatments, AXOS supplementation in young broilers was shown to stimulate or "kick-start" dietary AX digestion, thereby speeding up the development of a fiber-fermenting microbiome in the young broiler. This stimulation effect of AXOS could enable greater functional value to be extracted from dietary fiber in broiler feeds.

Lysolecithin, but not lecithin, improves nutrient digestibility and growth rates in young broilers.

1. The potential of lecithin and lysolecithin to improve lipid digestion and growth performance was investigated in three experiments: 1. an in vitro model that mimics the intestinal conditions of the chick, 2. a digestibility trial with chicks (5-7 days of age), and 3. a performance trial until 21 days of age. 2. In experiment 1, palm oil (PO), palm oil with lecithin (PO+L), and palm oil with lysolecithin (PO+LY) were subjected to in vitro hydrolysis and applied to Caco-2 monolayers to assess lipid absorption. 3. The in vitro hydrolysis rate of triglycerides was higher in PO+LY (k = 11.76 × 103/min) than in either PO (k = 9.73 × 103/min) or PO+L (k = 8.41 × 103/min), and the absorption of monoglycerides and free fatty acids was highest (P < 0.01) for PO+LY. In experiment 2, 90 broilers were assigned to three dietary treatments: a basal diet with 4% palm oil, and the basal diet supplemented with either 250 ppm lecithin or lysolecithin. 4. ATTD of crude fat was higher in broilers supplemented with lysolecithin, but was lower in broilers supplemented with lecithin. DM digestibility and AMEn in birds supplemented with lysolecithin were significantly higher (3.03% and 0.47 MJ/kg, respectively). 5. In experiment 3, 480 broilers were randomly allocated to four dietary treatments: basal diet with soybean oil (2%), basal diet with lecithin (2%), soybean oil diet with 250 ppm lysolecithin, or lecithin oil diet with 250 ppm lysolecithin. 6. Lecithin diets significantly reduced weight at day 10 and 21 compared with soybean oil. However, the addition of lysolecithin to lecithin-containing diets significantly improved bird performance. 7. The results of these studies showed that, in contrast to lecithin, lysolecithin was able to significantly improve the digestibility and energy values of feed in young broilers.

The effect of reduced balanced protein diet on the behavior of female broiler breeders in 2 generations.

The behavior of 2 generations of broiler breeders undergoing a 25% reduced balanced protein (RP) dietary treatment was investigated in the current study. There were 2 treatments for the F0 generation: control (C) breeders fed with standard C diets and RP breeders fed with RP diets. The female progeny of each treatment was again subjected to 2 dietary treatments, resulting in 4 treatments for F1 generation: C/C, C/RP, RP/C, and RP/RP (breeder feed in F0/F1 generation). To maintain the target body weights throughout the trial, breeders on RP diet received on average 10% more feed than C diet breeders. The behavior of the breeders at 8h30 (30 min before feeding at 9h00), 12h00, and 15h30 in weeks 23 and 37 of the F0 generation and in week 6, 11, and 22 of the F1 generation was observed. Litter scratching, feather pecking, and object pecking were occasionally increased by RP diet feeding which indicated feeding frustration. Drinking behavior decreased dramatically by the RP dietary feeding and resulting in a better litter condition which could benefit dust bathing behavior. In addition, feeding the breeders RP diet in the F0 generation decreased litter scratching (week 6) and feather pecking (week 22, 15h30) but increased sitting (week 11, 15h30) and drinking (a tendency in week 6 and a significant effect in week 11) behavior of offspring breeders (F1 generation). In general, breeders fed with reduced balanced protein diets, to some extent, spent less time drinking and their offspring could have an adaptation to the maternal RP diet. The mechanism of this adaptation still needs to be further investigated. In general, positive effects were found by reducing protein level of breeder diets. However, negative side effects such as feeding frustration were also observed, which merit further study.

Age-related arabinoxylan hydrolysis and fermentation in the gastrointestinal tract of broilers fed wheat-based diets.

Endoxylanases are frequently used in cereal-based broiler feeds to improve the nutritional quality of the feed. It is hypothesized that the age of broilers and the age-related development of their intestinal microbiota influence the efficacy of these enzymes. Hence, the objective of this study was to identify possible age-related changes in arabinoxylan (AX) digestion in the different parts of the gastrointestinal (GI) tract of broilers. A feeding trial was performed with 240 1-day-old chicks (Ross 308) receiving a wheat-based feed containing no supplemented endoxylanase. Digesta samples from every section of the GI tract were collected at 5, 10, 15, 21, 28, and 35 d of age and analyzed for AX content, AX digestibility, intestinal viscosity, and microbial endoxylanase and arabinofuranosidase activities. In the first 2 wk, the microbiota were able to solubilize a part of the water-unextractable arabinoxylan (WU-AX), thereby increasing intestinal viscosity and water-extractable arabinoxylan (WE-AX) concentrations in the GI tract. In these young birds, WU-AX and WE-AX with low arabinose to xylose ratios were able to enter the caeca but were not yet extensively fermented by the caecal microbiota as indicated by the high caecal AX concentrations at 5 and 10 d (P < 0.01). Establishment of a more mature microbial community at 3 wk of age resulted in a further increase in both the solubilization of WU-AX and fermentation of WE-AX at the ileum and caecum (P < 0.10). Furthermore, the increase in AX degrading enzyme activities with age denotes the high AX degrading capacity of the caecal microbiota. Finally, a total tract AX digestion of 24% was achieved at slaughter age (day 35). Our results clearly indicate that the capacity of intestinal microbiota to degrade AX in the hindgut increases as the broiler ages. This suggests that the benefits of endoxylanase supplementation of broiler feeds depend on the interaction of the intestinal microbiota and AX present in the GI tract at specific broiler ages.

Dual-energy X-ray absorptiometry is a reliable non-invasive technique for determining whole body composition of chickens.

In this study, a Lunar Prodigy dual-energy X-ray absorptiometry (DEXA) scanner was validated as a technique to estimate chicken body composition in a non-invasive way. Former research has emphasized the importance of validation of every scanner and software version. In a first trial, DEXA estimated body composition for broilers was correlated with chemical carcass analysis to develop prediction equations. As such, those equations can be used in later experiments with chickens to correct DEXA estimations to estimate body composition accurately by DEXA. DEXA estimated fat mass, lean tissue mass, bone mineral content (BMC) and total body mass, which is the sum of fat, lean mass and BMC, were compared to chemically analyzed crude fat, lean mass as the sum of protein and water and body ash content and scale body weight, respectively. Those regression equations were then used in a second trial to determine body composition based upon DEXA for breeders at different ages. In this experiment, fat and lean tissue determined by DEXA, were compared to dissection parameters commonly used for assessing carcass quality, namely breast muscle and abdominal fat. The first trial showed that DEXA provides high correlations for body mass (ρ = 1) and the individual tissue masses separately (ρ ranging between 0.98 and 1). These high correlations allow for accurate prediction of those components with the developed regression equations. Proportional fat and lean tissue were correlated with their chemical counterparts, however, to a lower extent than absolute values due to lower variation between the proportional weights. BMC percentage was not significantly correlated with ash percentage. Furthermore, in trial 2 high correlations were observed between dissection parameters and DEXA-corrected estimations. These correlations show that DEXA can assess carcass quality in breeders without sacrificing the birds. In conclusion, DEXA is a reliable technique to estimate breeder and broiler body composition in a non-invasive way, hence allowing for longitudinal studies over longer periods of time while avoiding sacrificing of birds.

The learning ability and memory retention of broiler breeders: 2 transgenerational effects of reduced balanced protein diet on reward-based learning.

The effect of reduced balanced protein (RP) diet in the F0 and F1 generation of broiler breeders on the learning ability and memory retention of the F2 generation was investigated by means of a reward v. no reward discrimination T-maze test. There were two treatments for the F0 generation: control (C) group, reared on standard commercial diets, and reduced balanced protein (RP) group, fed with RP diets (25% reduction in CP and amino acids). The female F0-progeny of each treatment was again separated into the two dietary treatments, resulting in four treatments for the F1 generation: C/C, C/RP, RP/C and RP/RP (breeder feed in F0/F1 generation). The RP diets fed breeders received on average 10% more feed than C diets fed breeders to achieve a similar target BW. The F2 generation was composed of four treatments coming from the female F1-progeny of the four treatments and were all fed with C diet (namely C/C/C, C/RP/C, RP/C/C and RP/RP/C). All four F2 generation groups were able to complete the T-maze learning test with a slight difference in success rate but a significant difference within groups was observed regarding the time needed to complete the test. In general, the RP/RP/C group needed more time for completing the test compared with the other three groups and the shortest time was recorded for the RP/C/C group. At similar ages, breeders with early learning experience spent significantly less time in completing the test compared with unexperienced breeders. Long-term memory retention was observed in all four groups whereas the learning ability in solving the test decreased with age. It took longer for the breeders to complete the test at older ages. In conclusion, under our experimental conditions, the RP dietary treatment in previous generations had no influence on the T-maze learning ability and memory retention of broiler breeders of the third generation, although it might have effects on the working performance in the T-maze learning test of F2 generation breeders.

The learning ability and memory retention of broiler breeders: 1 effects of reduced balanced protein diet on reward based learning.

Learning and mental abilities of farm animals are important for their adaptation to new environments and could serve as an indicator for welfare and performance. This study assessed the learning ability and memory retention of broiler breeders through a T-maze test with a reward v. no reward set-up. Feed supplemented with or without meal worms served as a reward. Two trials were conducted with either parent stock (PS) breeders or pure line A (PL) breeders and with the same dietary treatment namely a control (C) group fed with standard commercial diets and a reduced balanced protein (RP) group fed with RP diets (25% reduction of CP and amino acids). To maintain similar target BW, the RP group received on average 10% more feed. A tonic immobility (TI) test was performed to estimate the fearfulness of PS breeders. Most breeders were capable of completing the T-maze tests, but the C group needed less time compared with the RP group when their own feed was given as a reward. However, when meal worms were provided as an extra incentive, the RP group completed the maze significantly faster than the C group. Compared to the C breeders, the RP breeders remained longer in a TI state, indicating a higher level of fearfulness. Long-term memory retention in the T-maze test was observed in both groups. It is concluded that the RP diet had no influences on the learning ability of the reward v. no reward discrimination test and its memory retention of broiler breeders. The increased amount of time the RP breeders needed to solve the test was probably due to a higher sense of fear. Furthermore, the RP diet enhanced the motivation of breeders to obtain an alternative feed such as meal worms.

Multigenerational effects of a reduced balanced protein diet during the rearing and laying period of broiler breeders. 1. Performance of the F1 breeder generation.

Studies on mammals and poultry showed that maternal dietary treatments can alter the offspring performance. However, in contrast to rodent studies, little is known about multigenerational dietary manipulations in broiler breeders. The presented research aimed to investigate the effects of a reduction of 25% in the dietary crude protein (CP) level in the F0 generation on the body composition and reproductive performance of F1 broiler breeders. In the F0 generation, breeders were fed either a control (C) or reduced balanced protein (RP) diet, 25% reduction in crude protein and amino acids. Female F0-progeny of each treatment were fed a C or RP diet, resulting in 4 treatments in the F1 breeder generation: C/C, C/RP, RP/C, and RP/RP. The reproductive performance of breeders fed RP diets was negatively influenced by the dietary CP reduction in the F1 generation (P < 0.001). Moreover, breeders descending from hens that received RP diets in the F0 generation showed a significantly reduced reproductive capacity compared to their control fed counterparts (P < 0.001). Breeders fed RP diets in the F1 generation were characterized by higher plasma T3 concentrations (P < 0.001), an increased proportional abdominal fat pad (P < 0.001) and proportional liver weight (P < 0.001). During the rearing phase, the RP fed breeders needed a higher feed allowance, whereas no differences could be observed between the C/C and RP/C or the C/RP and RP/RP breeders. However, breeders originating from birds fed RP diets in the F0 generation needed lower feed allocations in the laying phase to maintain a similar body weight. Egg weight was reduced for the C/RP and RP/RP breeders. At 34 wk of age, eggs from C/RP and RP/RP breeders showed a reduced proportional albumen weight, whereas no effects on egg composition were found at 42 wk of age. It was concluded that prenatal protein undernutrition triggered hens to relocate more energy towards growth and maintenance and less towards reproductive capacity.

Multigenerational effects of a reduced balanced protein diet during the rearing and laying period of broiler breeders. 2. Zootechnical performance of the F1 broiler offspring.

Several studies in mammals focused on the maternal programming of the metabolism by epigenetic mechanisms, while currently, the consequences of a maternal dietary treatment on the offspring performance of farm animals are of particular interest for commercial purpose. In the present study, we investigated if the zootechnical performance of the progeny was altered by a maternal dietary treatment, being a lower dietary crude protein (CP) of the grandparent and/or parent generation. The multigenerational effects of a reduced maternal CP content were investigated by reducing the dietary CP level by 25% in rearing and laying diets of pure line A breeders. The F0 generation breeders were fed either control (C) or reduced balanced protein (RP) diets. The F1 breeder generation was constructed by dividing the F0 female progeny again over a C or RP diet, resulting in 4 dietary treatments in the F1 generation: C/C, C/RP, RP/C, and RP/RP (letters indicating the diets in, respectively, F0 and F1 generations). The offspring performance was evaluated by a zootechnical and nitrogen retention trial on C and low-protein (LP) broiler diets. For the C broiler diet, the C/RP and RP/RP offspring were characterized by a higher BW from d 35 until d 42 compared to the C/C progeny, whereas the RP/C offspring had an intermediate BW that did not differ from the other groups. A tendency (P = 0.067) towards a better nitrogen retention was observed for the offspring of breeders that received the RP diets in F0 and/or F1 generation compared to the C/C progeny. For the LP broiler diet, the C/RP (P = 0.021) and RP/C (P = 0.001) offspring had a higher BW compared to the C/C progeny during the entire grow-out period. In addition, the C/RP offspring were characterized by a lower FCR from d 28 onwards (P = 0.021). In conclusion, dietary treatments imposed on mother hens can have direct effects on the next generation, as well as indirect effects on multiple generations.

Effect of different split-feeding treatments on performance, egg quality, and bone quality of individually housed aged laying hens.

In order to try to combat the effect of age on eggshell quality in aged laying hens, 5 split-feeding treatments were compared with conventional feeding between 75 and 92 wk of age. In the conventional treatment (T1), the same morning (M) and afternoon (A) diet was fed containing fine (FL) and coarse (CL) limestone at a 50:50 ratio. In the split treatments, the ratio of FL and CL was 50:50 or 30:70, and time of administration (M/A) differed. The following treatments were given: T2 = 50FL-M:50CL-A, T3 = 50CL-M:50FL-A, T4 = 30FL-M:20FL-A+50CL-A, T5 = 30FL-M:70CL-A, and T6 = 0M:30FL-A+70CL-A. A total of 12 individually housed Dekalb white hens was used per treatment. Feed intake, nutrient intake, and laying % was lower in T1 compared to all split treatments (P ≤ 0.001). Due to this low performance in T1, split feeding could not be compared to the conventional system in this trial. By the end of the trial, eggshell quality was improved in T1 as a result of low laying % and more frequent and longer laying pauses. In the split treatments, laying % and feed intake were similar, except in T3 in which a decrease was observed after 81 wk (P ≤ 0.05). Egg weight was higher in T5 and T6 due to higher total and morning protein intake compared to T3 (P ≤ 0.05). Feeding only 50FL-A in T3 not only resulted in lower performance but also in consistently lower shell thickness, indicating a negative effect of suboptimal limestone supplementation. In the split-feeding system, the most optimal combination of morning and afternoon diet was a morning diet with only FL and an afternoon diet with only CL (T2), which both provided ∼50% of the total daily Ca intake. Shell breaking strength and dynamic stiffness could be maintained on this diet between 75 and 92 weeks. Decreasing the amount of Ca in the morning and increasing it in the afternoon did not improve shell quality traits. Bone quality was not affected by limestone particle size or inclusion level in the split-feeding system.

The effects of a reduced balanced protein diet on litter moisture, pododermatitis and feather condition of female broiler breeders over three generations.

Protein content reduction in broiler breeder diets has been increasingly investigated. However, broiler breeders reared on low protein diets are characterized by a deterioration of the feather condition. Furthermore, polydipsia induced by controlled feed intake increases litter moisture and as a consequence pododermatitis. This project aimed to study the litter moisture, pododermatitis and feather condition of breeders fed with a 25% reduced balanced protein (RP) diet during the rearing and laying period over three successive generations. The experiment started with two treatments for the F0 generation: control (C) group fed with standard C diets and RP group fed with RP diets. The female F0-progeny of each treatment was divided into the two dietary treatments as well, resulting in four treatments for the F1 generation: C/C, C/RP, RP/C and RP/RP (breeder feed in F0/F1 generation). The RP diet fed breeders received on average 10% more feed than C diet fed breeders to achieve the same target BW. The female F1-progeny of each treatment were all fed with C diets which resulted in four treatments for the F2 generation: C/C/C, C/RP/C, RP/C/C and RP/RP/C (breeder feed in F0/F1/F2 generation). Litter moisture, footpad and hock dermatitis were recorded at regular intervals throughout the experimental period in all three generations. For the F0 and F1 generation, the pens of breeders receiving C diets had significantly higher litter moisture than the RP diets fed groups (P<0.05), resulting in an elevated footpad dermatitis occurrence (FDO) (P<0.05). No difference was found in the F2 generation. The feather condition was scored during the laying period for each generation. F0 and F1 breeders reared on the RP diets had poorer feather condition than those receiving the C diets (P<0.05). The C/RP breeders had a significantly poorer feather condition than RP/RP breeders (P<0.05). For the F2 generation, RP/RP/C breeders had a significantly better feather condition compared with the other three groups (P<0.05). The RP/C/C breeders were significantly better feathered than C/C/C breeders (P<0.05). In conclusion, providing RP diets to broiler breeders improved litter condition and hence reduced FDO whereas impaired feather condition. Furthermore, positive transgenerational effects of the maternal RP diets on the feather condition may be inferred, hence potentially altering the welfare status.

Feeding broiler breeders a reduced balanced protein diet during the rearing and laying period impairs reproductive performance but enhances broiler offspring performance.

Mammalian studies have shown that nutritional constraints during the perinatal period are able to program the progeny (metabolism, performance). The presented research aimed to investigate if broiler breeders and their offspring performance could be influenced by reducing the dietary crude protein (CP) level with 25%. A total of 160 day-old pure line A breeder females were randomly divided over 2 dietary treatments. The control group was fed commercial diets, whereas the reduced balanced protein (RP) breeders received an isoenergetic diet that was decreased with 25% in dietary CP and amino acid during their entire lifespan. The RP birds required an increased feed allowance, varying between 3 and 15%, to meet the same BW goals as their control fed counterparts. The difference in feed allocations and reduction of the dietary CP level resulted in a net protein reduction varying between 14 and 23%. At wk 27 and 40, the body composition of the breeders was changed as a result of the dietary treatment. At both ages, the proportional abdominal fat pad weight of the RP breeders was increased (P < 0.001), whereas the proportional breast muscle weight was only higher at wk 27 in the control group compared to the RP group (P < 0.001). Egg weight (P < 0.001) and egg production (P < 0.001) was decreased for the RP fed birds. The lower dietary CP level reduced the proportional albumen weight of the RP eggs (P = 0.006). Male offspring from RP breeders were characterized by an increase in BW from 28 d until 35 d of age (P = 0.015). Moreover, female progeny of RP breeders showed a reduced FCR (P = 0.025), whereas male progeny showed a tendency (P = 0.052) towards a lower FCR at 5 wk of age. In conclusion, lowering dietary CP levels in rearing and laying phase of breeders had a negative effect on breeder performance but enhanced live performance of the offspring.

Supplementation of fine and coarse limestone in different ratios in a split feeding system: Effects on performance, egg quality, and bone strength in old laying hens.

Different ratios of fine- (FL) and coarse limestone (CL) were compared (50FL:50CL, 30FL:70CL, 0FL:100CL) in conventional (C) and split feeding (S) for their effects on performance, egg quality traits, and bone quality of old, brown laying hens (72 to 83 wk). Each treatment consisted of 42 hens (7 hens × 6 replicates). In the C system diets supplemented with limestone were provided during the whole day, whereas in the S system a morning diet was fed without added limestone, and only the afternoon diet was supplemented with different limestone ratios. Highest laying %, egg mass, and lowest feed conversion were found in the C system with 50FL:50CL and 0FL:100CL and in the S system with 30FL:70CL between 76 and 79 wk (P ≤ 0.001). Reduced cracked egg % was found when 0FL:100CL was supplemented in the C system and 30FL:70CL in the S system between 72 and 83 wk (P ≤ 0.001). Tibia ash content was higher in the S system compared to the C system (P = 0.005); tibia breaking strength, however, did not differ between feeding systems. Egg quality traits were not improved by S feeding. However, at 83 wk, S feeding resulted in higher breaking strength, but lower shell thickness compared to the C system (P = 0.036, P ≤ 0.001, respectively). Therefore, hens in the S feeding system might have been able to form a structurally superior shell compared to the C system. For further investigation, instead of restricting limestone supplementation solely to the afternoon, it might be a better approach to provide FL and CL at a different time of the d in a split feeding system to improve shell quality in old, brown laying hens in an extended production cycle.

Changes in egg quality traits during the last phase of production: is there potential for an extended laying cycle?

1. A field study was conducted to investigate the deterioration of eggshell quality at the end of lay and examine the potential to extend the production cycle. A total of 1200 eggs were assessed originating from commercial farms in Belgium: eggs were random sampled at the collection belt at 57-65 weeks of age and at the end of the production cycle (74-92 weeks). 2. Based on a linear mixed model, egg quality was estimated for an average flock at 60 weeks of age and changes in quality traits were predicted until the end of the laying cycle. 3. Egg weight increased by 0.07 g every week from 60 weeks of age onwards and shape index decreased by 0.04 unit weekly (P < 0.001). Haugh unit values decreased weekly by 0.38 units whereas relative yolk weight was unaffected after 60 weeks of age. Relative albumen weight showed a weekly increase of 0.02% and relative shell decreased by 0.02% weekly until the end of lay. 4. Shell quality traits were also influenced by age: shell index decreased by 0.013 g weekly, shell thickness was calculated to be 0.23 µm thinner every week, deformation increased by 0.06 µm weekly from 60 weeks to the end of the laying cycle. Variability of the deformation of eggs indicated more heterogeneous shell quality at the end of the production cycle. Dynamic stiffness increased from 60 weeks of age on. 5. Laying hens were depopulated on average at 80 weeks of age (varying from 74-92 weeks). Although ageing had a significant effect on most of the egg quality traits, egg quality was still acceptable at the end of lay, indicating the potential to extend the laying cycle.