Characterization of Growth and Reproduction Performance,Transgene Integration,Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer

Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance and characterized the transgene insertion, transmission, and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs. Multiple copies of monogenic EGFP transgene were inserted at noncoding sequences of host genome, and passed from founder transgenic pigs to their transgenic offspring in segregation or linkage manner. The EGFP transgene was ubiquitously expressed in transgenic pigs, and its expression intensity was associated with transgene copy number but not related to its promoter DNA methylation level. To the best of our knowledge, this is first study that fully described the growth and reproduction performance, transgene insertion, expression, and transmission profiles in transgenic pigs produced by piggyBac system. It not only demonstrates that piggyBac transposition-mediated gene transfer is an effective and favorable approach for pig transgenesis, but also provides scientific information for understanding the transgene insertion, expression and transmission patterns in transgenic animals produced by piggyBac transposition.     

‘Tailception’: using neural networks for assessing tail lesions on pictures of pig carcasses

Tail lesions caused by tail biting are a widespread welfare issue in pig husbandry. Determining their prevalence currently involves labour intensive, subjective scoring methods. Increased societal interest in tail lesions requires fast, reliable and cheap systems for assessing tail status. In the present study, we aimed to test the reliability of neural networks for assessing tail pictures from carcasses against trained human observers. Three trained observers scored tail lesions from automatically recorded pictures of 13 124 pigs. Nearly all pigs had been tail docked. Tail lesions were classified using a 4-point score (0=no lesion, to 3=severe lesion). In addition, total tail loss was recorded. Agreement between observers was tested prior and during the assessment in a total of seven inter-observer tests with 80 pictures each. We calculated agreement between observer pairs as exact agreement (%) and prevalence-adjusted bias-adjusted κ (PABAK; value 1=optimal agreement). Out of the 13 124 scored pictures, we used 80% for training and 20% for validating our neural networks. As the position of the tail in the pictures varied (high, low, left, right), we first trained a part detection network to find the tail in the picture and select a rectangular part of the picture which includes the tail. We then trained a classification network to categorise tail lesion severity using pictures scored by human observers whereby the classification network only analysed the selected picture parts. Median exact agreement between the three observers was 80% for tail lesions and 94% for tail loss. Median PABAK for tail lesions and loss were 0.75 and 0.87, respectively. The agreement between classification by the neural network and human observers was 74% for tail lesions and 95% for tail loss. In other words, the agreement between the networks and human observers were very similar to the agreement between human observers. The main reason for disagreement between observers and thereby higher variation in network training material were picture quality issues. Therefore, we expect even better results for neural network application to tail lesions if training is based on high quality pictures. Very reliable and repeatable tail lesion assessment from pictures would allow automated tail classification of all pigs slaughtered, which is something that some animal welfare labels would like to do.     

The effects of immunization against gonadotropin-releasing hormone on growth performance,reproductive activity and carcass traits of heavy weight gilts

Heavy weight gilts commonly show signs of oestrus during the late finishing phase, which results in a period of reduced feed intake and growth rate. Immunization against gonadotropin-releasing hormone (GnRH) (IM, immunocastration) was developed for finishing boars and recently extrapolated to females. Immunocastration acts by suppressing reproductive activity and improving the growth potential. The objective of this study was to evaluate the effects of IM on growth performance, reproductive activity and carcass characteristics of late finishing gilts. Seventy-two gilts (63.49 ± 0.39 kg) were either injected with saline (Intact) or immunized against GnRH (Immunized). The study consisted of three experimental periods: between the first to second immunization (V1 to V2, 15 to 19 weeks of age), from the second immunization to the beginning of daily boar exposure (DBE) (V2 to DBE, 19 to 21 weeks of age) and from the beginning of DBE to slaughter (S) (DBE to S, 21 to 25 weeks of age). Immunized gilts showed an overall increase (from 15 to 25 weeks) of 3.90 kg (P < 0.05) of live weight, 56 g (P < 0.05) of average daily gain (ADG) and 250 g (P < 0.001) of average daily feed intake (ADFI). Immunized gilts had a greater ADFI (+240 g, P < 0.05) and worse feed conversion ratio (+0.26, P < 0.05) from 19 (V2) to 21 weeks of age (before DBE). Furthermore, those females had higher feed intake (+410 g; P < 0.001) plus greater daily weight gain (+92 g; P < 0.05) from V2 to S, and from DBE to S (+470 g of ADFI, P < 0.001; +129 g of ADG, P < 0.01, respectively). Immunocastration had no effect on backfat thickness, lean meat percentage and weight, cold carcass yield or loin depth (P > 0.05). Immunized gilts showed 4.4% increased cold carcass weight (P < 0.01) and 10.6% greater gross flank weight (P < 0.001). Immunization against GnRH did not influence shoulder, collar, loin, belly or ham weights. Nor did it influence belly fat thickness, or meat, skin plus fat and bones yields of cold ham (P > 0.05). Immunocastration reduced ovarian and uterine weights by 82% (P < 0.001) and 93% (P < 0.001), respectively, and suppressed oestrus manifestation in all gilts in the immunized group (P < 0.001). These results indicate that immunization against GnRH is a promising tool for stimulating growth performance with no detrimental effects on carcass quality of heavy weight finishing gilts, by means of oestrus suppression.     

Standardized total tract digestible phosphorus requirement of 6 to 13 kg pigs fed diets without or with phytase

Dietary phosphorus concentration greatly affects pig’s growth performance, environmental impact and diet cost. A total of 1080 pigs (initially 5.9 ± 1.08 kg) from three commercial research rooms were used to determine the effects of increasing standardized total tract digestible (STTD) P concentrations in diets without and with phytase on growth performance and percentage bone ash. Pens (10 pigs/pen, 9 pens/treatment) were balanced for equal weights and randomly allotted to 12 treatments. Treatments were arranged in two dose titrations (without or with 2000 units of phytase) with six levels of STTD P each. The STTD P levels were expressed as a percentage of NRC (2012) requirement estimates (% of NRC; 0.45 and 0.40% for phases 1 and 2, respectively) and were: 80%, 90%, 100%, 110%, 125% and 140% of NRC in diets without phytase and 100%, 110%, 125%, 140%, 155% and 170% of NRC in diets with phytase. Diets were provided in three phases, with experimental diets fed during phases 1 (days 0 to 11) and 2 (days 11 to 25), followed by a common diet from days 25 to 46. On day 25, radius samples from one median-weight gilt per pen were collected for analysis of bone ash. During the treatment period, increasing STTD P from 80% to 140% of NRC in diets without phytase improved average daily gain (ADG; quadratic, P < 0.01), average daily feed intake (ADFI; quadratic, P < 0.05) and gain–feed ratio (G : F; linear, P < 0.01). Estimated STTD P requirement in diets without phytase was 117% and 91% of NRC for maximum ADG according to quadratic polynomial (QP) and broken-line linear (BLL) models, respectively, and was 102%, 119% and >140% of NRC for maximum G : F using BLL, broken-line quadratic and linear models, respectively. When diets contained phytase, increasing STTD P from 100% to 170% of NRC improved ADG (quadratic, P < 0.05) and G : F (linear, P < 0.01). Estimated STTD P requirement in diets containing phytase was 138% for maximum ADG (QP), and 147% (QP) and 116% (BLL) of NRC for maximum G : F. Increasing STTD P increased (linear, P < 0.01) the percentage bone ash regardless of phytase addition. When comparing diets containing the same STTD P levels, phytase increased (P < 0.01) ADG, ADFI and G : F. In summary, estimated STTD P requirements varied depending on the response criteria and statistical models and ranged from 91% to >140% of NRC (0.41% to >0.63% of phase 1 diet and 0.36% to >0.56% of phase 2 diet) in diets without phytase, and from 116% to >170% of NRC (0.52% to >0.77% of phase 1 diet and 0.46% to >0.68% of phase 2 diet) for diets containing phytase. Phytase exerted an extra-phosphoric effect on promoting pig’s growth and improved the P dose-responses for ADG and G : F.     

Effect of the inclusion of food waste in pig diets on growth performance, carcass and meat quality

Food waste from fish and fruit shops was used as an alternative to the grain in grower-finisher pig diets. Two diets were formulated on an iso-nutrient basis (14 MJ digestible energy per kg, 160 g crude protein per kg on a dry-matter basis) to contain 0 g of food waste per kg in the control diet and 50 g of fish-shop waste per kg and 120 g of fruit-shop waste per kg in the experimental diet. In the study, 28 pigs per diet (seven pigs per pen) were fed ad libitum from 20 kg to 100 kg, then, they were slaughtered and the carcass characteristics determined. The inclusion of food waste in the diet had no significant effect (P>0.05) on average daily feed intake (2.12 v. 2.20 kg/day), average daily gain (0.74 v. 0.78 kg/day), or gain/feed (0.35 v. 0.35 kg per kg). In the case of the experimental diet, backfat thickness was significantly lower (18.0 v. 21.3 mm, P < 0.01). The results of the taste test indicated that the meat from food waste-fed pigs had acceptable organoleptic quality although a very light aroma to fish was observed in the bacon (P < 0.01).It was concluded that food waste from the fish and fruit shops could be included in grower-finisher pig diets without any detrimental effect on growth performance and only minor effects on carcass characteristics and meat quality.     

True phosphorus digestibility and the endogenous phosphorus outputs associated with brown rice for weanling pigs measured by the simple linear regression analysis technique

The objectives of this study were to determine true phosphorus (P) digestibility, degradability of phytate-P complex and the endogenous P outputs associated with brown rice feeding in weanling pigs by using the simple linear regression analysis technique. Six barrows with an average initial body weight of 12.5 kg were fitted with a T-cannula and fed six diets according to a 6 × 6 Latin-square design. Six maize starch-based diets, containing six levels of P at 0.80, 1.36, 1.93, 2.49, 3.04, and 3.61 g/kg per kg dry-matter (DM) intake (DMI), were formulated with brown rice. Each experimental period lasted 10 days. After a 7-day adaptation, all faecal samples were collected on days 8 and 9. Ileal digesta samples were collected for a total of 24 h on day 10. The apparent ileal and faecal P digestibility values of brown rice were affected ( P < 0.01) by the P contents in the assay diets. The apparent ileal and faecal P digestibility values increased from − 48.0 to 36.7% and from − 35.6 to 40.0%, respectively, as P content increased from 0.80 to 3.61 g/kg DMI. Linear relationships ( P < 0.05), expressed as g/kg DMI, between the apparent ileal and faecal digestible P and dietary levels of P, suggested that true P digestibility and the endogenous P outputs associated with brown rice feeding could be determined by using the simple regression analysis technique. There were no differences ( P>0.05) in true P digestibility values (57.7 ± 5.4 v. 58.2 ± 5.9%), phytate P degradability (76.4 ± 6.7 v. 79.0 ± 4.4%) and the endogenous P outputs (0.812 ± 0..096 v. 0.725 ± 0.083 g/kg DMI) between the ileal and the faecal levels. The endogenous faecal P output represented 14 and 25% of the National Research Council (1998) recommended daily total and available P requirements in the weanling pig, respectively. About 58% of the total P in brown rice could be digested and absorbed by the weanling pig. Our results suggest that the large intestine of the weanling pigs does not play a significant role in the digestion of P in brown rice. Diet formulation on the basis of total or apparent P digestibility with brown rice may lead to P overfeeding and excessive P excretion in pigs.     

Pig acute-phase protein levels after stress induced by changes in the pattern of food administration

A total of 240 pigs, 74 days old, half boars and half females, were included in a trial designed to assess the effect of the stress caused by changes in the pattern of food administration on the concentration of acute phase proteins (APP) and productive performance parameters. Half of the animals (pigs fed ad libitum, AL group) had free access to feed, while the rest were fed following a disorderly pattern (DIS group), in which animals had alternating periods of free access to feed and periods of no feeding, when food was removed from the feeder. The periods of free access to feed (two daily periods of 2-h duration) were randomly assigned, and varied from day to day. Total feed supplied per day was identical in both groups, and exceeded the minimal amount required for animals of these ages. Pen feed intake, individual body weights and the main positive pig APP pig major acute phase protein (Pig-MAP), haptoglobin, serum amyloid A (SAA), C-reactive protein (CRP), and the negative APP apolipoprotein A-I (ApoA-I) and transtherytin were determined every 2 weeks during the period 76 to 116 days of age. Animals fed ad libitum had better average daily gain (ADG) than DIS animals in the whole experimental period (P < 0.01) but the differences in ADG were only produced in the two first experimental sub-periods (60 to 74 and 74 to 116 days of age), suggesting that the stress diminished when the animals get used to the DIS feeding. Interestingly differences in ADG between DIS and AL pigs were due to males, whereas no differences were observed between females. The same differences observed for ADG were found for APP. DIS males had higher Pig-MAP concentration than AL males at 74 and 116 days of age, lower ApoA-I concentration at 74 days of age and higher haptoglobin and CRP concentration at 116 days of age (P < 0.05). The results obtained in this trial show an inverse relationship between weight gain and APP levels, and suggest that APP may be biomarkers for the evaluation of distress and welfare in pigs.     

Effects of fermented oyster mushroom ( Pleurotus ostreats) by-product supplementation on growth performance, blood parameters and meat quality in finishing Berkshire pigs

The objective of the present study was to investigate the effects of fermented oyster mushroom (Pleurotus ostreats) by-production (FOMP) supplementation on the growth performance, blood parameters, carcass traits and meat quality in finishing Berkshire pigs. FOMP was made by mixing oyster mushroom by-production with rice bran and barley bran and this mixture was fermented for 60 days. The experimental diets were 0, 3, 5 and 7% of FOMP added to C, T1, T2 and T3 in the basis diet for 7 weeks. Average daily gain (kg/day) was higher in C and T1 than in T2 and T3 ( P < 0.05). Average daily feed intake (kg/day) and feed conversion increased by the addition of FOMP ( P < 0.05). Total cholesterol and high-density lipoprotein cholesterol were higher in T3 than other treatments ( P < 0.05). Carcass weight (kg) was higher in C and T1 than in T2 and T3 ( P < 0.05). Dressing (%) was higher in C than in T3 ( P < 0.05). Crude protein was lower in T3 than in other treatments ( P < 0.05). Crude fat was higher in T2 and T3 than in C ( P < 0.05). pH₂₄ was higher in C than in other treatments ( P < 0.05). Cooking loss (%) was higher in T1 than T2 ( P < 0.05). Water-holding capacity (%) was higher in C than in T1 ( P < 0.05). In meat colour, CIE a* was lower by the addition of FOMP ( P < 0.05). CIE b* was higher in C than in other treatments ( P < 0.05). In backfat colour, CIE L* was lower in T3 than other treatments ( P < 0.05). CIE b* was lower by addition of FOMP ( P < 0.05). Palmitoleic and oleic acid were higher in T3 than in other treatments ( P < 0.05). Linoleic and arachidonic acids were higher in T2 than in other treatments ( P < 0.05). The results indicate that 3% of FOMP affected the growth performance, carcass traits, meat quality and fatty acid in contrast to addition of 5% of FOMP for Berkshire pigs during the finishing period.     

Effects of dietary incorporation of different antioxidant extracts and free-range rearing on fatty acid composition and lipid oxidation of Iberian pig meat

This investigation was designed to evaluate the effects of feeding either free range or in confinement using concentrated diets with the same ingredients and oil source (5.5% of olive oleins) but with different antioxidant supplementation [control diet with a basal level of α-tocopheryl acetate (control); 200 mg/kg synthetic all-rac-α-tocopheryl acetate (Eall-rac); 200 mg/kg natural RRR-α-tocopheryl-acetate (ERRR-); flavonoid extract-enriched diet (AFlav); and phenolic compound-enriched extract (APhen)] on the fatty acid composition and lipid oxidation of Iberian pig muscle longissimus dorsi. The α-tocopherol concentration was significantly higher in muscles from free-range and ERRR- pigs than in muscles from Eall-rac pigs, and γ-tocopherol was only detected in muscles from free-range pigs. Longissimus dorsi muscles from free-range pigs had a significantly lower content of saturated fatty acids and higher content of polyunsaturated fatty acids than muscles from the other five groups of pigs fed in confinement; however, no significant effect on monounsaturated fatty acids was observed. No effect of dietary antioxidant supplementation (synthetic or natural α-tocopherol, flavonoid extract, or phenol extract) on the fatty acid composition of muscles was observed. A significant influence of dietary treatment on lipid oxidation was observed after 3 (P < 0.01), and 7 and 10 (P < 0.001) days of refrigerated storage, respectively. The lowest thiobarbituric acid-reactive substances (TBARS) values were found in pork chops from the free-range and ERRR- groups, intermediate values from the Eall-rac group, followed by AFlav and APhen, while the highest TBARS values corresponded to muscles from pigs fed the control concentrate. The source of α-tocopherol had a significant effect on lipid oxidation (P < 0.05), whereas the AFlav and APhen groups had similar TBARS values.