Weaning differentially affects mitochondrial function, oxidative stress, inflammation and apoptosis in normal and low birth weight piglets.

Weaning is associated with increased occurrence of infections and diseases in piglets. Recent findings indicate that weaning induces mitochondrial dysfunction and oxidative stress conditions that more severely impact smaller piglets. The objective of this study was to characterize the molecular mechanisms underlying these physiological consequences and the relation with systemic inflammatory status in both normal and low birth weight (NBW and LBW) piglets throughout the peri-weaning period. To conduct the study, 30 sows were inseminated, and specific piglets from their litters were assigned to one of two experimental groups: NBW (n = 60, 1.73 ± 0.01 kg,) and LBW piglets weighing less than 1.2 kg (n = 60, 1.01 ± 0.01 kg). Then, 10 piglets from each group were selected at 14, 21 (weaning), 23, 25, 29 and 35 days of age to collect organ and plasma samples. Specific porcine RT2 Profiler™ PCR Arrays related to mitochondrial function, oxidative stress, inflammation and apoptosis processes were first used to target genes that are modulated after weaning in NBW piglets (d 23 and d 35 vs. d 14). Expression of selected genes was evaluated by quantitative PCR. These analyses revealed that expression of inflammatory genes CXCL10 and CCL19 increased after weaning in intestinal mucosa, while expression of genes encoding subunits of the mitochondrial respiratory chain was downregulated in liver and kidney of both groups. Interestingly, major modulators of mitophagy (BNIP3), cell survival (BCL2A1) and antioxidant defense system (TXNRD2, GPx3, HMOX1) were found to be highly expressed in NBW piglets. The systemic levels of TNF-α and IL1-ß significantly increased following weaning and were higher in NBW piglets. These results provide novel information about the molecular origin of mitochondrial dysfunction and oxidative stress observed in weaned piglets and suggest that clearance of dysfunctional mitochondria, antioxidant defenses and inflammatory response are compromised in LBW piglets.

Growing Pigs' Interest in Enrichment Objects with Different Characteristics and Cleanliness.

Enrichment objects can be a practical way to provide rooting and chewing material to growing pigs, on which they can express species-specific behaviors. The challenge is to provide enrichment objects that will satisfy pigs' behavioral needs, while being practical and low-cost for the producers. Two trials were conducted to evaluate the effects of object characteristics such as design, location, cleanliness or degree of wear, on pigs' interest over time. The first trial compared seven objects, varying in their design and location, presented individually for five consecutive days to groups of 12 ± 3 (average ± SD) pigs, weighing 61 ± 9.2 kg. The pigs' interest in the objects was evaluated based on the frequency, total duration and mean length of manipulation with the objects. All objects were manipulated at different levels depending on their characteristics. On average, the pigs interacted more frequently (p < 0.001) with a chewable object made of three polyurethane balls, spring-mounted and anchored to the floor, and spent more time manipulating a dried wood beam on the floor (p < 0.05), which was destructible and chewable, than suspended ropes, plastics and rubber objects, and a plastic ball on the floor. The second trial used two-choice preference tests to compare objects varying in their degree of cleanliness or wear, presented in pairs to growing pigs weighing 47 ± 7 kg and housed in groups of 14 ± 1. Two identical objects were placed simultaneously in a pen over 5 days, and only one of them was cleaned or replaced daily (treatment) while the duplicate was left untouched (control). The results showed no clear preference between control and treatment objects, indicating that short-term maintenance of the objects might be unnecessary.

Modelling piglet growth and mortality on commercial hog farms using variables describing individual animals, litters, sows and management factors.

Increases in sow prolificacy have reduced piglet vitality, growth capacity and weight at weaning and even pig weight at slaughter. The aim of this study was to develop a model that predicts likelihood of mortality and weight at weaning. A database containing 3214 records of birth weight, weight gain at 24h, rectal temperature at 24h, litter size, age at weaning, fostering status, manual assistance of birth and oxytocin use as well as the corresponding 227 records of sow parity and feed intake was analysed using logit functions for mortality and linear functions for weaning weight. The best model of mortality predicted increased likelihood as birth weight, rectal temperature and 0-24h weight gain decreased and sow parity and time between births increased (P<0.01, χ2=2910). The best model of weaning weight predicted increases with increasing birth weight, 0-24h body weight gain, age at weaning and sow parity and decreases with increasing litter size at 24h (P<0.01; AICC=4324; RMSE=0.82). This study confirmed that birth weight and weight gain during the first 24h are the principal factors influencing piglet growth and pre-weaning mortality.

A preliminary study of the effects of individual response to challenge tests and stress induced by humans on learning performance of weaned piglets (Sus scrofa).

This study investigated whether individual behavioural characteristics of piglets and stress induced by experience with humans can influence learning performance. After weaning, piglets received a chronic experience with humans to modulate their emotional state: rough (ROU), gentle (GEN), or minimal (MIN) experience. Simultaneously, they were trained on a discrimination task. Afterward, their behaviour during challenge tests was assessed. The first learning step of the task involved associating a positive sound cue with a response (approach a trough) and success of piglets depended mostly on motivation to seek for reward. Although the experience with humans did not have direct effect, the degree of fear of handler, measured based on their reactivity to a human approach test, was related to motivation to seek rewards and learning speed of this first step in stressed ROU piglets, but not in MIN and GEN piglets. In contrast, the second learning step was more cognitively challenging, since it involved discrimination learning, including negative cues during which piglets had to learn to avoid the trough. Locomotion activity, measured during an open-field test, was associated with performance of the discrimination learning. To conclude, fearfulness towards humans and locomotion activity are linked with learning performance in relation to task complexity, highlighting the necessity to take into account these factors in animal research and management.

The Way Humans Behave Modulates the Emotional State of Piglets.

The emotional state can influence decision-making under ambiguity. Cognitive bias tests (CBT) proved to be a promising indicator of the affective valence of animals in a context of farm animal welfare. Although it is well-known that humans can influence the intensity of fear and reactions of animals, research on cognitive bias often focusses on housing and management conditions and neglects the role of humans on emotional states of animals. The present study aimed at investigating whether humans can modulate the emotional state of weaned piglets. Fifty-four piglets received a chronic experience with humans: gentle (GEN), rough (ROU) or minimal contact (MIN). Simultaneously, they were individually trained on a go/no-go task to discriminate a positive auditory cue, associated with food reward in a trough, from a negative one, associated with punishments (e.g. water spray). Independently of the treatment (P = 0.82), 59% of piglets completed the training. Successfully trained piglets were then subjected to CBT, including ambiguous cues in presence or absence of a human observer. As hypothesized, GEN piglets showed a positive judgement bias, as shown by their higher percentage of go responses following an ambiguous cue compared to ROU (P = 0.03) and MIN (P = 0.02) piglets, whereas ROU and MIN piglets did not differ (P > 0.10). The presence of an observer during CBT did not modulate the percentage of go responses following an ambiguous cue (P > 0.10). However, regardless of the treatment, piglets spent less time in contact with the trough following positive cues during CBT in which the observer was present than absent (P < 0.0001). This study originally demonstrates that the nature of a chronic experience with humans can induce a judgement bias indicating that the emotional state of farm animals such as piglets can be affected by the way humans interact with them.

The perception of humans by piglets: recognition of familiar handlers and generalisation to unfamiliar humans.

Humans are part of the environment of domestic animals and interact with them daily, providing a good basis for the study of interspecific relationships. Abilities to discriminate and recognise individuals form the basis of these relationships, and they are crucial skills for domestic animals, since individual humans can differ in their behaviour towards them. At the same time, with experience, animals may develop a general memory of humans and generalise their behaviour towards strangers. This study aimed to determine the extent to which weaned piglets can discriminate familiar humans and generalise their past experience when faced with unfamiliar humans. Forty-eight groups of three piglets were submitted to two consecutive 5-day conditioning periods of the same or opposite valence (positive and/or negative) given by one (A) or two (A then B) handlers. The reactivity of piglets to a motionless human and to an approaching human was measured before and after conditioning periods with unfamiliar and familiar handlers. Thereafter, piglets which received treatments with the two handlers A and B were subjected to a choice test between both handlers. The reactivity to an approaching human appeared to better reflect the past experience with familiar handlers since piglets clearly recognised them and adapted their behaviour accordingly. In contrast, reaction of piglets to a motionless human reflected their natural propensity to seek interaction, even after a negative experience. Although piglets were able to extend their memory of humans to an unfamiliar human through generalisation, many factors seemed to interact in this process, especially the complexity of the previous experience (inconsistent vs consistent) and the context in which piglets met the unfamiliar human (motionless vs approaching). We conclude that discrimination and generalisation processes of reactivity to humans do not simply rely on past interactions, but also depend on the context according to the degree of similitude with their past experience.

Welfare of Pigs Being Transported over Long Distances Using a Pot-Belly Trailer during Winter and Summer.

A total of 2,145 pigs were transported for 8 h in summer (six trips) and winter (five trips) using a pot-belly trailer accommodating pigs in four locations (upper deck or UD, bottom-nose or BN, middle deck or MD and bottom deck or BD). Heart rate of pigs during loading and transportation and lactate and creatine kinase (CK) concentrations in exsanguination blood were measured. Meat quality was evaluated in the Longissimus thoracis (LT), Semimembranosus (SM) and Adductor (AD) muscles. During summer, pigs loaded in the UD and MD had higher (P < 0.05) heart rate at loading compared to those located in the BD and BN. Blood lactate and CK concentrations were higher (P < 0.001) in winter than in summer. Lactate concentration was higher (P = 0.01) in the blood of pigs transported in the BN. Pigs transported in the BN had higher pHu values in the LT, SM and AD muscles (P = 0.02, P < 0.001 and P = 0.002, respectively) and lower (P = 0.002) drip loss values in the SM muscle. This study confirms that some locations within the PB trailer have a negative impact on the welfare of pigs at loading and during transport with more pronounced effects in the winter due to the additive effect of cold stress.

Fish oil diets alter the phospholipid balance, fatty acid composition, and steroid hormone concentrations in testes of adult pigs.

The objective was to determine the effect of long-term dietary supplementation of two types of fish oil on lipid composition and steroidogenesis in adult pig testis. Twenty-four Duroc boars, aged 204.5 ± 9.4 d (body weight 128.1 ± 16.7 kg) received daily 2.5 kg of an iso-caloric basal diet supplemented with: 1) 62 g of hydrogenated animal fat (AF); 2) 60 g of menhaden oil (MO) containing 16% of eicosapentaenoic acid (EPA) and 18% of docosahexaenoic acid (DHA); or 3) 60 g of tuna oil (TO) containing 7% of EPA and 33% of DHA. After these diets were consumed for 7 mo, testicular hormones, phospholipid content, and fatty acid composition of individual phospholipids in testis were determined. Body and reproductive organ weights were not significantly affected by dietary treatments. Testicular tissue from boars fed a TO diet, followed by those receiving MO and AF diets, had the lowest level of phosphatidylethanolamine (TO < MO < AF; P < 0.01) but the highest sphingomyelin (TO > MO > AF; P < 0.01). For each phospholipid, boars fed either the MO or TO diet had increased total omega-3 fatty acids, particularly DHA (P < 0.01), by reciprocal replacement of total omega-6 fatty acids (20:4n-6, 22:5n-6). The MO diet increased EPA more than the other diets. Testicular concentrations of testosterone and estradiol were lower in boars fed a TO diet than a MO diet (P < 0.02). In conclusion, long-term dietary supplementation of fish oil, regardless of the EPA/DHA ratio, modified the fatty acid compositions in testis and affected steroid production of healthy adult boars, which may represent a promising models for future studies on fertility.

Fish oil diets do not improve insulin sensitivity and secretion in healthy adult male pigs.

The effects of long-term dietary supplementation of fish oil (n-3 PUFA-rich) in adult male pigs on body condition as well as insulin sensitivity and secretion were examined. Fifteen Duroc boars aged 204.5 (sd 9.4) d (body weight 145.8 (sd 16.8) kg) received daily 2.5 kg basal diet with a supplement of: (1) 62 g hydrogenated animal fat (n 5); (2) 60 g menhaden oil containing 10.8 g DHA and 9.0 g EPA (n 6); (3) 60 g tuna oil containing 19.8 g DHA and 3.9 g EPA (n 4). Rations were balanced to be isoenergetic. After 7 months of treatments, oral glucose and meal tolerance tests were conducted after insertion of a catheter into the jugular vein. Dietary supplementation with n-3 PUFA altered the blood plasma profile: DHA and EPA increased whereas arachidonic acid decreased (P < 0.01). Plasma glucose, insulin and C-peptide responses to oral glucose and the test meal were not affected by treatments (P>0.34). For all animals, total body fat estimated from body weight and back fat thickness was correlated with both beta-cell function (by homeostasis model assessment (HOMA); r+0.63) and insulin sensitivity (index of whole-body insulin sensitivity and by HOMA; r - 0.63 and r+0.66, respectively). In conclusion, long-term supplementation with dietary n-3 PUFA did not affect insulin metabolism in healthy adult male pigs. The relationship between body fat and insulin sensitivity, well documented in human subjects, suggests that the adult male pig could be a promising animal model for studies on insulin metabolism.

Effect of an environmentally relevant metabolized organochlorine mixture on porcine cumulus-oocyte complexes.

Organochlorine compounds and their metabolites bioaccumulate and have been detected in follicular and genital tract fluids of humans and animals. This study was designed to investigate the effect of a metabolised organochlorine mixture, extracted from plasma of sows treated with an environmentally relevant organochlorine mixture in the course of a previous study, on porcine cumulus-oocyte complexes (COCs) in vitro. The major component of the metabolised mixture is 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE) at 15.1 mg/l, which accounts for 40.7% of the total extract. Polychlorinated biphenyls (PCBs) account for 30.8% of the extract and hydroxylated PCB metabolites (OH-PCBs) for 11.8%. Exposure of COCs to the metabolised mixture induced a decrease of apoptotic cumulus cells at low concentrations and an increase at higher concentrations following a U-shaped curve (p=0.0106), with the intermediate treatment (3.6 microg/l OH-PCBs) significantly reducing apoptosis compared to the extraction control (p=0.05). However, the metabolised mixture did not affect cumulus expansion, oocyte maturation, penetration, development to blastocyst, or the number of cells per blastocyst. This study also indicates that organochlorine metabolites similar in concentrations to levels found in Arctic populations can affect growing cumulus-oocyte complexes without inducing an overt toxicological response.

Effect of folic acid plus glycine supplement on uterine prostaglandin and endometrial granulocyte-macrophage colony-stimulating factor expression during early pregnancy in pigs.

The objective was to determine the effects of folic acid+glycine supplement on uterine metabolism of prostaglandin and mRNA expression of endometrial granulocyte-macrophage colony-stimulating factor (GM-CSF) in nulliparous (NYL) and multiparous Yorkshire-Landrace (YL) sows, and in multiparous Meishan-Landrace sows (ML). In each of these three groups, sows were randomly assigned to two treatments: 15 ppm folic acid+0.6% glycine or no supplement. The dietary supplement was given from the estrus before mating to slaughter on Day 25 of pregnancy. At slaughter, endometrial tissue was collected to determine endometrial expression levels of GM-CSF mRNA, cyclooxygenase-1 (COX1) and -2 (COX2) and to evaluate in vitro endometrial secretion of prostaglandin E2 (PGE2) secretion. Allantoic fluid samples were also collected to determine the concentration of PGE2, prostaglandin F2alpha (PGF2alpha), estradiol-17beta (E2), progesterone (P4), and transforming-growth factor-beta2 (TGF-beta2). The allantoic contents of PGF2alpha, E2 and P4, and endometrial in vitro secretion of PGE2 were not significantly influenced by the folic acid+glycine supplement. The folic acid+glycine supplement tended (P<0.07) to increase allantoic content of PGE2 and TGF-beta2 in all sows and increased (P<0.05) endometrial expression of COX2, especially in NYL sows. The endometrial expression of COX1 was decreased (P<0.05) by folic acid+glycine supplement, especially in multiparous YL sows. The allantoic contents of PGE2 and PGF2alpha were not significantly affected by sow type. However, NYL sows had higher (P<0.05) endometrial in vitro secretion of PGE2 and allantoic content of P4 than multiparous YL and ML sows. The allantoic content of E2 was also higher (P<0.05) in NYL sows than in multiparous ML sows only. The allantoic content of TGF-beta2 was lower (P<0.05) in multiparous ML than in multiparous YL only sows. Finally, in YL and NYL sows, folic acid+glycine supplement decreased (P<0.05) the endometrial expression of GM-CSF but not in ML sows. In summary, folic acid+glycine supplement altered endometrial expression of GM-CSF and uterine metabolism of prostaglandins during the post-attachment period of porcine embryos but some of these effects were manifest only in Meishan and nulliparous sows.

Effects of breed, parity, and folic Acid supplement on the expression of folate metabolism genes in endometrial and embryonic tissues from sows in early pregnancy.

Folic acid and glycine are factors of great importance in early gestation. In sows, folic acid supplement can increase litter size through a decrease in embryonic mortality, while glycine, the most abundant amino acid in the sow oviduct, uterine, and allantoic fluids, is reported to act as an organic osmoregulator. In this study, we report the characterization of cytoplasmic serine hydroxymethyltransferase (cSHMT), T-protein, and vT-protein (variant T-protein) mRNA expression levels in endometrial and embryonic tissues in gestating sows on Day 25 of gestation according to the breed, parity, and folic acid + glycine supplementation. Expression levels of cSHMT, T-protein, and vT-protein mRNA in endometrial and embryonic tissues were performed using semiquantitative reverse transcription-polymerase chain reaction. We also report, for the first time, an alternative splicing event in the porcine T-protein gene. Results showed that a T-protein splice variant, vT-protein, is present in all the tested sow populations. Further characterizations revealed that this T-protein splice variant contains a coding intron that can adopt a secondary structure. Results demonstrated that cSHMT mRNA expression levels were significantly higher in sows receiving the folic acid + glycine supplementation, independently of the breed or parity and in both endometrial and embryonic tissues. Upon receiving the same treatment, the vT-protein and T-protein mRNA expression levels were significantly reduced in the endometrial tissue of Yorkshire-Landrace sows only. These results indicate that modulation of specific gene expression levels in endometrial and embryonic tissues of sows in early gestation could be one of the mechanism involved with the role of folic acid on improving swine reproduction traits.

Effects of folic acid and vitamin B12 supplements on folate and homocysteine metabolism in pigs during early pregnancy.

The present experiment aimed to determine the effects of supplements of folic acid (FA) alone or in combination with vitamin B12 on folate and homocysteine metabolism in gestating nulliparous Yorkshire-Landrace (YL) and multiparous Landrace (LD) occidental sows and multiparous Chinese Meishan-Landrace (ML) sows. LD sows were randomly assigned to two treatments: 0 or 15 mg FA/kg diet while YL and ML sows were assigned to three treatments: 0 mg FA/kg diet, 15 mg FA/kg or 15 mg vitamin B12/kg diet. Supplements were given from the oestrus preceding insemination up to slaughter on day 15 of gestation. At slaughter, a uterine flush was collected to determine uterine contents of homocysteine, methionine, tetrahydrofolate (THF), 5-methyl-THF, pyridoxal 5-phosphate (P5P) and vitamin B12. Blood samples were taken at first oestrus, at insemination and on days 5, 10 and 15 of gestation to determine plasma concentrations of homocysteine, methionine, THF, 5-methyl-THF, P5P, vitamin B12 and relative total folate-binding capacity. In occidental sows (YL and LD), the FA supplement tended to decrease uterine flush content of homocysteine (P=0.06) and concentrations of plasma homocysteine (P=0.09). Nulliparous YL sows had lower concentrations of plasma homocysteine, methionine, THF and 5-methyl-THF (P<0.05) than multiparous LD sows. Multiparous ML and LD sows had similar concentrations of plasma THF, 5-methyl-THF, methionine and vitamin B12, but ML sows had lower concentrations of plasma homocysteine (P<0.05). The vitamin B12 supplement increased concentrations of plasma vitamin B12 (P<0.05) both in multiparous ML and nulliparous YL sows, but had no effect on the composition of either uterine flush or plasma. The present results showed also that sows had a low vitamin B12 status (<200 pg/ml) and high circulating homocysteine levels (>15 microm) during the first 15 d of gestation. Furthermore, the vitamin B12 content in uterine secretions represented between 180 and 300 % of the total content in plasma. The low plasma concentrations of homocysteine in multiparous ML sows suggest a more efficient remethylation pathway which may not be dependent upon dietary supply of FA or vitamin B12. In nulliparous YL sows, low concentrations of both homocysteine and methionine suggest that the methionine requirement for protein deposition might have reduced the amount of methionine available for the methylation pathway. The results of the present experiment suggest that the reduction of uterine homocysteine may be an important aspect of the role of FA supplement on the uterine environment in occidental sows. The presence of high levels of vitamin B12 in uterine secretions merits further investigation in relation to embryonic development.