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Background/objectives: Saliva is gaining importance as a diagnostic sample in pigs. The aim of this research was to evaluate a panel of salivary analytes in three porcine diseases and establish predictive models to detect them., Methods: Saliva samples were obtained from healthy pigs ( n = 97) and pigs affected by meningitis due to Streptococcus suis ( n = 118), diarrhea due to enterotoxigenic Escherichia coli (ETEC, n = 77), and porcine reproductive and respiratory syndrome (PRRS, n = 52). The following biomarkers were analyzed: adenosine deaminase (ADA), haptoglobin (Hp), calprotectin (Calp), aldolase, alpha-amylase (sAA), lactate dehydrogenase (LDH), total protein (TP), and advanced oxidation protein products (AOPPs). Predictive models based on binary logistic regression and decision trees combining those analytes for detecting specific diseases were constructed., Results: The results showed a different biomarker profile between the groups. S. suis and ETEC pigs showed higher values of ADA, Hp, Calp, aldolase, sAA, LDH, and TP than healthy pigs. Pigs with PRRS showed higher values of Hp, Calp, sAA, and LDH than healthy animals. The constructed predictive models showed overall accuracies of over 78% and 87% for differentiating ETEC and PRRS, respectively, whereas the models did not accurately predict S. suis infection., Conclusions: Salivary analytes show different changes in pigs depending on the disease, and the combination of these analytes can contribute to the prediction of different diseases. Further studies should be conducted in larger populations to confirm these findings and evaluate their possible practical applications.

Adequate nutritional management in maternities is one of the most challenging aspects of swine production. This study reports the results of two studies aiming to determine, under commercial farm conditions, the effects of precision feeding (electronic sow feeders, ESFs) on the production and economic yields of lactating sows and possible nutritional and metabolic differences when compared to a control group fed with traditional feeders. The first trial showed that sows fed with ESFs weaned heavier piglets than sows fed with traditional feeders. Feed intake during the lactation period was similar in the sows of both groups; consequently, the amount of feed per kg of weaned piglet was lower in the sows fed with ESFs, which is a remarkable economic output. The second trial confirmed these findings and showed that, despite similar feed intakes, the sows fed with ESFs had lower bodyweight losses during the lactation period, but there were no major differences in milk composition or metabolic traits of sows and piglets.

Antioxidant supplementation in critical periods may be useful for improvement of piglet early viability and development. We have evaluated the effects of maternal perinatal diet inclusion of a high vitamin E level (VE, 100 mg all-rac-α-tocopheryl acetate /kg), hydroxytyrosol (HT, 1.5 mg/kg), or their combination (VEHT), in comparison to a control diet (C, 30 mg all-rac-α-tocopheryl acetate /kg), on the offspring homeostasis and metabolism, analysing the weaned piglets' adipose tissue transcriptome and adipocyte morphology. Diets were provided to pregnant Iberian sows (n = 48, 12 per treatment) from gestation day 85 to weaning (28 days postpartum) and 48 piglets (n = 12 per treatment) were sampled 5 days postweaning for dorsal subcutaneous adipose tissue analyses. RNA obtained from 6 animals for each diet was used for paired-end RNA sequencing. Results show that supplementation of sows' diet with either vitamin E or hydroxytyrosol had substantial effects on weaned piglet adipose transcriptome, with 664 and 587 genes being differentially expressed, in comparison to C, respectively (q-value<0.10, Fold Change>1.5). Genes upregulated in C were mainly involved in inflammatory and immune response, as well as oxidative stress, and relevant canonical pathways and upstream regulators involved in these processes were predicted as activated, such as TNF, IFNB or NFKB. Vitamin E, when supplemented alone at high dose, activated lipid biosynthesis functions, pathways and regulators, this finding being accompanied by increased adipocyte size. Results suggest an improved metabolic and antioxidant status of adipose tissue in animals born from sows supplemented with individual antioxidants, while the combined supplementation barely affected gene expression, with VEHT showing a prooxidant/proinflamatory functional profile similar to C animals. Different hypothesis are proposed to explain this unexpected result. Findings allow a deeper understanding of the processes taking place in adipose tissue of genetically fat animals and the role of antioxidants in the regulation of fat cells function., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Laviano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cystatin C, ammonia, and bicarbonate have been described to be biomarkers of sepsis and inflammation in humans. The saliva of pigs can be used to detect a wide range of pathogens but also many biomarkers that can be analyzed to evaluate different conditions such as stress (i.e., cortisol and alpha amylase), immune system (i.e., ADA, S100 proteins), inflammation (i.e., acute phase proteins), redox status (i.e., various antioxidants and oxidants), and general metabolism or the status of different organs and tissues. However, there is a lack of assays for the possible measurement and use of cystatin C, ammonia, and bicarbonate in saliva as biomarkers of sepsis or inflammation in pigs. The objective of this study was to validate commercially available automated assays for the measurement of cystatin C, ammonia, and bicarbonate in the saliva of pigs, having the advantage of using a noninvasive sample that is easy to collect. The assays were precise and accurate, and the recommended storage condition for the saliva samples was -80 °C. In addition, cystatin and ammonia showed significant increases in the saliva of pigs with S. suis infection, whereas bicarbonate decreased. Further studies would be recommended to increase knowledge about the possible potential applications of the measurements of these three analytes in the saliva of pigs as biomarkers to evaluate the animals' health and welfare.

Pig feeding prior to the extensive fattening phase might affect the final lipid profile and product quality. This study evaluates how maternal supplementation with vitamin E (VITE) (100 mg/kg), hydroxytyrosol (HXT) (1.5 mg/kg), or combined administration (VE + HXT) affects the piglet's plasma and tissues' fatty acid profiles and lipid stability according to the sow's parity number (PN), as well as the possible changes to the lipid profile after extensive feeding. The sows' PN affected the total fatty acid profile of plasma, muscle, and liver of piglets, with lower Δ-9 and Δ-6 desaturase indices but higher Δ-5 in those from primiparous (P) than multiparous (M) sows. Dietary VITE was more effective at decreasing C16:0 and saturated fatty acids in the muscle of piglets born from M than P sows, and modified the liver phospholipids in a different way. Sows' supplementation with HXT increased C18:2n-6 in triglycerides and polyunsaturated fatty acids (PUFA) in muscle phospholipids. In the liver, HXT supplementation also increased free-PUFA and free-n-3 fatty acids. However, lipid oxidation of piglets' tissues was not affected by the antioxidant supplementation, and it was higher in the livers of piglets born from M sows. The fatty acid profile in the muscle of pigs after extensive feeding was not affected by the PN, but it was by the sows' antioxidant supplementation, with positive effects on quality by both compounds.

An assay for the measurement of myeloperoxidase (Mpx) in porcine saliva was developed and validated, and factors influencing Mpx and another two biomarkers of inflammation and immune system, the protein S100A12 and the inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), were studied. The spectrophotometric method for Mpx measurement validated in this assay showed an adequate analytical performance including precision and accuracy. When a group of twenty healthy pigs was sampled every 4 h from 8 a.m. until 8 p.m., Mpx and S100A12 showed significant increases at 4 p.m., whereas ITIH4 concentration showed a significant decrease at 12 a.m. Increases were also seen in salivary Mpx, S100A12, and ITIH4 levels 24 h after the intramuscular administration of Escherichia coli lipopolysaccharide in five pigs; whereas in a non-septic inflammation after the subcutaneous administration of turpentine oil to five pigs changes were seen in S100A12 at 3 h and in ITIH4 at 48 h. When a stressful situation consisting of the transportation and stay of 4 h to a slaughterhouse of 24 pigs was performed, all analytes were increased after 4 h of lairage in the slaughterhouse compared with the values that were obtained the day before at the same time of the day. Mpx can be measured in the saliva of pigs with the automated assay described in this report. Mpx, S100A12, and ITIH4 salivary levels can change depending on the hour of the day in which the sample is taken, and increases can be produced due to sepsis, non-septic inflammation and stress., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests or potential conflicts of interest with respect to the research, authorship, and/or publication of this article. None of the authors has any financial or personal relationships that could inappropriately influence the content of the paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

An adequate intestinal environment before weaning may contribute to diarrhea predisposition and piglet development. This study evaluates how the dietary supplementation of vitamin E (VE) (100 mg/kg), hydroxytyrosol (HXT) (1.5 mg/kg) or the combined administration (VE + HXT) given to Iberian sows from gestation affects the piglet's faecal characteristics, short chain fatty acids (SCFAs), fatty acid profile or intestinal morphology as indicators of gut health; and quantify the contribution of the oxidative status and colostrum/milk composition to the piglet's SCFAs content and intestinal health. Dietary VE increased isobutyric acid (iC4), butyric acid (C4), isovaleric acid (iC5), and ∑SCFAs, whereas HXT increased iC4 and tended to decrease ∑SCFAs of faeces. Piglets from HXT-supplemented sows also tended to have higher faecal C20:4n-6/C20:2 ratio C22:6 proportion and showed lower occludin gene expression in the duodenum. The combination of both antioxidants had a positive effect on iC4 and iC5 levels. Correlation analyses and regression equations indicate that faecal SCFAs were related to oxidative status (mainly plasma VE) and colostrum and milk composition (mainly C20:2, C20:3, C20:4 n-6). This study would confirm the superiority of VE over HXT supplementation to improve intestinal homeostasis, gut health, and, consequently piglet growth.

Maternal nutrition during gestation plays an important role in colostrum production, postnatal growth, and survival of newborn lambs, especially in twin gestations. This research aimed to investigate the effects of chronic natural undernutrition on colostrum traits and early lamb's postnatal growth born from single and twin sheep pregnancies developed in a restrictive prairie, representative of southern Patagonia. Single- and twin-bearing ewes ( n  = 20 per group) were maintained grazing in a natural pasture. At 140 days of gestation, ewes were placed in individual pens for lambing control. Colostrum was collected immediately after delivery and at 12, 24, and 36 h postpartum, for determination of yield and composition. Maternal blood was obtained at 140 days of gestation and at lambing for plasma glucose, progesterone, 17β-estradiol, and IgG determination. Newborn lamb blood for determining glycaemia and IgG was collected at birth and at 12, 24, 36, and 120 h after birth. Lamb mortality and growth was assessed from birth until 30 days of life. No differences were observed in progesterone and 17β-estradiol. There were no differences in colostrum yields and fat components, however single- had higher values of protein and lactose than twin-bearing ewes ( p < 0.05 for both). Singletons had higher glycaemia than twins at 12 h postpartum (102.2 ± 32.8 vs. 73.4 ± 29.9 mg/dL, p < 0.05 ). Colostrum IgG content was similar at delivery but higher in single ewes at 12 and 24 h, reaching a similar values at 36 h (4.7 ± 9.7 and 5.8 ± 7.7 mg/mL in single and twin pregnancies, respectively). Newborn IgG was higher in singletons compared to twins at least until 48 h of life. Lams body weight was always superior in singleton than twins from birth until 30 days of life. Mortality did not differ during the first week of life, but it increased significantly only in twins until day 30 of life. Undernourishment in pregnant ewes affected colostrum quantity and quality, resulting in a lower postnatal growth and a higher mortality in twins. Alternative managements favoring fetal growth, birth weight and neonatal viability in twin sheep pregnancies are needed, when flocks are breed under harsh environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Turín, Sales, Peralta, De los Reyes, Borie, Carrasco, González-Bulnes and Parraguez.)

The family of calgranulins includes S100A8 (calgranulin A), S100A9 (calgranulin B), which can appear as a heterodimer known as S100A8/A9 or calprotectin, and S100A12 (calgranulin C). These proteins are related to different inflammatory conditions, immune-mediated diseases, and sepsis and are considered biomarkers of potential interest. This study aims to evaluate if S100A8/A9 and A12 could change in pigs with diarrhea due to E. coli and to compare the changes of S100A8/A9 and A12 with other analytes in order to explore the possible causes or mechanisms involved. For this purpose, a panel integrated by analytes related to inflammation (haptoglobin, inter-alpha trypsin inhibitor 4 (ITIH4), and total protein); immune system (adenosine deaminase, ADA); stress (alpha-amylase); tissue damage (lactate and lactate dehydrogenase (LDH)); sepsis (aldolase) and redox status (ferric-reducing ability of saliva (FRAS) and advanced oxidation protein products (AOPP)) was evaluated. S100A8/A9 and A12 and the other analytes measured in this study showed increases in the saliva of pigs with diarrhea due to E. coli . S100A8/A9 and/or A12 showed a significant correlation of different magnitude with some of the other analytes evaluated. Further studies should be conducted to gain knowledge about the possible practical applications as biomarkers of the measurements of S100A8/A9 and A12 in the saliva of pigs.

Background: Streptococcus suis (S. suis) is a Gram-positive bacteria that infects pigs causing meningitis, arthritis, pneumonia, or endocarditis. This increases the mortality in pig farms deriving in severe economic losses. The use of saliva as a diagnostic fluid has various advantages compared to blood, especially in pigs. In this study, it was hypothesized that saliva could reflect changes in different biomarkers related to stress, inflammation, redox status, and muscle damage in pigs with S. suis infection and that changes in these biomarkers could be related to the severity of the disease., Results: A total of 56 growing pigs from a farm were selected as infected pigs (n = 28) and healthy pigs (n = 28). Results showed increases in biomarkers related to stress (alpha-amylase and oxytocin), inflammation (haptoglobin, inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), total protein, S100A8-A9 and S100A12), redox status (advanced oxidation protein producs (AOPP)) and muscle damage (creatine kinase (CK), CK-MB, troponin I, lactate, aspartate aminotransferase, and lactate dehydrogenase). An increase in adenosine deaminase (ADA), procalcitonin, and aldolase in infected animals were also observed, as previously described. The grade of severity of the disease indicated a significant positive correlation with total protein concentrations, aspartate aminotransferase, aldolase, and AOPP., Conclusions: This report revealed that S. suis infection caused variations in analytes related to stress, inflammation, redox status, and muscle damage in the saliva of pigs and these can be considered potential biomarkers for this disease., (© 2023. The Author(s).)

Different feeding strategies are being applied to sows in order to obtain homogeneous piglets' weights and improved health status. This study evaluated how the dietary supplementation of vitamin E (VE) (100 mg/kg), hydroxytyrosol (HXT) (1.5 mg/kg) or the combined administration (VE + HXT) given to Iberian sows from day 85 of gestation affected the growth pattern of the piglets and their oxidative status; and quantified what these effects were due to. Dietary VE and HXT improved the oxidative status of sows and piglets. Both VE and HXT modified the growth pattern at birth and performances of the piglets in a different way according to the growing period. Piglets' performances were positively correlated with plasma VE and negatively with plasma malondialdehyde (MDA) of the sow. However, the highest variation in growth patterns was explained by the colostrum composition. Significant linear equations were observed between piglets' performances and colostrum saturated (SAT), n-7 monounsaturated fatty acids (C16:1n-7 and C18:1n-7) and different desaturases indices. This study would confirm that VE supplementation to the sow diet could be more adequate than HXT for the improved development during the first weeks of a piglet's life. The combined administration of both antioxidants would not produce additional positive effects compared to the individual supplementation.

Modifying the composition of a sow's milk could be a strategy to improve the intestinal health and growth of her piglet during the first weeks of life. This study evaluated how dietary supplementation of vitamin E (VE), hydroxytyrosol (HXT) or VE+HXT given to Iberian sows from late gestation affected the colostrum and milk composition, lipid stability and their relationship with the piglet's oxidative status. Colostrum from VE-supplemented sows had greater C18:1n-7 than non-supplemented sows, whereas HXT increased polyunsaturated (∑PUFAs), ∑n-6 and ∑n-3 fatty acids. In 7-day milk, the main effects were induced by VE supplementation that decreased ∑PUFAs, ∑n-6 and ∑n-3 and increased the Δ-6-desaturase activity. The VE+HXT supplementation resulted in lower desaturase capacity in 20-day milk. Positive correlations were observed between the estimated mean milk energy output and the desaturation capacity of sows. The lowest concentration of malondialdehyde (MDA) in milk was observed in VE-supplemented groups, whereas HXT supplementation increased oxidation. Milk lipid oxidation was negatively correlated with the sow's plasma oxidative status and to a great extent with the oxidative status of piglets after weaning. Maternal VE supplementation produced a more beneficial milk composition to improve the oxidative status of piglets, which could promote gut health and piglet growth during the first weeks, but more research is needed to clarify this.

Intrauterine Growth Restriction (IUGR) hinders the correct growth of the fetus during pregnancy due to the lack of oxygen or nutrients. The developing fetus gives priority to brain development ("brain sparing"), but the risk exists of neurological and cognitive deficits at short or long term. On the other hand, diets rich in fat exert pernicious effects on brain function. Using a pig model of spontaneous IUGR, we have studied the effect on the adult of a long-term high-fat diet (HFD) on the neurotransmitter profile in several brain areas, and the morphology and the proteome of the hippocampus. Our hypothesis was that animals affected by IUGR (born with low birth weight) would present a different susceptibility to an HFD when they become adults, compared with normal birth-weight animals. Our results indicate that HFD affected the serotoninergic pathway, but it did not provoke relevant changes in the morphology of the hippocampus. Finally, the proteomic analysis revealed that, in some instances, NBW and LBW individuals respond to HFD in different ways. In particular, NBW animals presented changes in oxidative phosphorylation and the extracellular matrix, whereas LBW animals presented differences in RNA splicing, anterograde and retrograde transport and the mTOR pathway.

Intrauterine Growth Restriction (IUGR) is a major problem in pig production and different strategies, mainly maternal supplementation with different agents, are currently being studied. The combination of hydroxytyrosol and n3-PUFA seems to be a promising treatment to counteract IUGR, since the combination may help improve n3-PUFA composition and lower the inflammatory status of IUGR piglets. The aim of the present study is to determine the effects of a maternal supplementation, from day 35 to day 100 of pregnancy, with linseed oil and hydroxytyrosol on the fetal FA composition. The results showed higher n3 levels, including eicosapentaenoic and docosahexaenoic FA in the offspring from treated gilts, which showed lower n6-PUFA/n3-PUFA (n6/n3) ratios. Saturated and monounsaturated fatty acids were also affected by treatment, especially in the muscle and brain. Thus, a maternal supplementation with linseed oil and hydroxytyrosol affected the fetal FA tissue composition, which could have implications in pig production due to the improvement of the piglets' health status.

The hypothalamus is implicated in controlling feeding and adiposity, besides many other physiological functions, and thus can be of great importance in explaining productive differences between lean and fatty pig breeds. The present study aimed to evaluate the hypothalamic transcriptome of pure Iberian (IBxIB) and Large White x Iberian crossbreds (IBxLW) at 60 days-old, produced in a single maternal environment. Results showed the implication of gender and genotype in the hypothalamic transcriptome, with 51 differentially expressed genes (DEGs) between genotypes and 10 DEGs between genders. Fourteen genotype by sex interactions were found, due to a higher genotype effect on transcriptome found in males. In fact, just 31 DEGs were identified when using only females but 158 using only males. A higher expression of genes related to mitochondrial activity in IBxIB male animals (ND3, ND4, ND5, UQCRC2 and ATP6) was found, which was related to a higher oxidative phosphorylation and greater reactive oxygen species and nitric oxide production. IBxLW male animals showed higher expression of SIRT3 regulator, also related to mitochondrial function. When females were analysed, such differences were not found, since only some differences in genes related to the tricarboxylic acid cycle. Thus, the results indicate a significant effect and interaction of the breed and the sex on the hypothalamic transcriptome at this early age., Competing Interests: The authors have declared that no competing interests exist.

Supplementation of a mother's diet with antioxidants such as hydroxytyrosol (HTX) has been proposed to ameliorate the adverse phenotypes of foetuses affected by intrauterine growth restriction (IUGR). Our previous studies showed, in a porcine model of IUGR, an effect of maternal HTX supplementation on the neurotransmitter profile of several brain areas and the morphology of the hippocampus in 100 days old foetuses. The present study analyzed the impact of maternal HTX supplementation on the hippocampus proteome at this foetal age by TMT10plex labelling. Eleven differentially abundant proteins were identified by comparing both conditions, and eight of them downregulated and three upregulated in the HTX-treated group. The downregulated proteins were mainly involved in protein synthesis and RNA metabolism and may explain the differences in neuron differentiation in the HTX-treated group. The upregulated proteins were related to cell detoxification and could represent a potential mechanism to explain the neuroprotective effect of HTX.

Intrauterine growth restriction (IUGR) refers to poor growth of a fetus during pregnancy due to deficient maternal nutrition or oxygen supply. Supplementation of a mother's diet with antioxidants, such as hydroxytyrosol (HTX), has been proposed to ameliorate the adverse phenotypes of IUGR. In the present study, sows were treated daily with or without 1.5 mg of HTX per kilogram of feed from day 35 of pregnancy (at 30% of the total gestational period), and fetuses were sampled at day 100 of gestation. Fetuses were classified as normal body weight (NBW) or low body weight (LBW) as a consequence of IUGR, constituting four groups: NBW-Control, NBW-HTX, LBW-Control, and LBW-HTX. The brain was removed, and the hippocampus, amygdala, and prefrontal cortex were rapidly dissected. Neuronal markers were studied by immunohistochemistry, and a decrease in the number of mature neurons in the hippocampal Cornu Ammonis subfield 1 (CA1) and the Dentate Gyrus (DG) regions was observed in LBW fetuses together with a higher number of immature neurons and other alterations in neuronal morphology. Furthermore, IUGR conditions altered the neurotransmitter (NT) profile, since an increase in the serotonin (5-HT) pathway was observed in LBW fetuses. Supplementation with HTX was able to reverse the morphological and neurochemical changes, leading both characteristics to values similar to those of NBW fetuses.

Maternal supplementation with antioxidants and n-3 PUFAs may be a promising strategy to reduce the risk of intrauterine growth restriction and preterm delivery, which may diminish the appearance of low-birth-neonates. A previous studies showed beneficial outcomes of the combination of hydroxytyrosol and linoleic acid, but there is no data of its prenatal effects. The present study aimed to determine the possible prenatal implications of such maternal supplementation at prenatal stages in swine, a model of IUGR pregnancies. Results showed effects on litter size, with treated sows having larger litters and, therefore, smaller fetuses. However, the brain/head weight ratio showed a positive effect of the treatment in development, as well as in some other major organs like lungs, spleen, or kidneys. On the other hand, treated piglets showed better glycemic and lipidemic profiles, which could explain postnatal effects. However, further research on the implications of the treatment on litter size and prenatal and postnatal development must be done before practical recommendation can be given.

Supplementation of a mother's diet with antioxidants, such as hydroxytyrosol (HTX), has been proposed to ameliorate the adverse phenotypes of fetuses at risk of intrauterine growth restriction. In the present study, sows were treated daily with or without 1.5 mg of HTX per kilogram of feed from day 35 of pregnancy (at 30% of total gestational period), and individuals were sampled at three different ages: 100-day-old fetuses and 1-month- and 6-month-old piglets. After euthanasia, the brain was removed and the hippocampus, amygdala, and prefrontal cortex were dissected. The profile of the catecholaminergic and serotoninergic neurotransmitters (NTs) was characterized and an immunohistochemical study of the hippocampus was performed. The results indicated that maternal supplementation with HTX during pregnancy affected the NT profile in a brain-area-dependant mode and it modified the process of neuron differentiation in the hippocampal CA1 and GD areas, indicating that cell differentiation occurred more rapidly in the HTX group. These effects were specific to the fetal period, concomitantly with HTX maternal supplementation, since no major differences remained between the control and treated groups in 1-month- and 6-month-old pigs.

The effects of a combined supplementation with herbal antioxidants during pregnancy on reproductive traits and piglet performance (number of live, dead, and mummified newborns and litter weight at birth and individual body weight at both birth and weaning) were assessed in a total of 1027 sows (504 treated and 523 control females) kept under commercial breeding conditions. The supplementation increased the number of live-born piglets (13.64 ± 0.11 vs. 12.96 ± 0.13 in the controls; p = 0.001) and the total litter weight, decreasing the incidence of low-weight piglets without affecting the number of stillbirths and mummified newborns. Such an effect was modulated by the number of parity and the supplementation, with supplementation increasing significantly the number of living newborns in the first, second, sixth, and seventh parities (0.87, 1.10, 1.49, and 2.51 additional piglets, respectively; p < 0.05). The evaluation of plasma vitamin concentration and biomarkers of oxidative stress (total antioxidant capacity, TAC, and malondialdehyde concentration, MDA) performed in a subset of farrowing sows and their lighter and heavier piglets showed that plasma levels of both vitamins were significantly higher in the piglets than in their mothers ( p < 0.05 for vitamin C and p < 0.005 for vitamin E), with antioxidant supplementation increasing significantly such concentrations. Concomitantly, there were no differences in maternal TAC but significantly higher values in piglets from supplemented sows ( p < 0.05). On the other hand, supplementation decreased plasma MDA levels both in the sows and their piglets ( p < 0.05). Finally, the piglets from supplemented mothers showed a trend for a higher weaning weight ( p = 0.066) and, specifically, piglets with birth weights above 1 kg showed a 7.4% higher weaning weight ( p = 0.024). Hence, the results of the present study, with high robustness and translational value by offering data from more than 1000 pregnancies under standard breeding conditions, supports that maternal supplementation with herbal antioxidants during pregnancy significantly improves reproductive efficiency, litter traits, and piglet performance.

We tested whether periconceptional nutrition with cladodes from the cactus, Opuntia ficus-indica, with or without protein-enrichment, improved the metabolomic profile and reproductive outcomes of adult female sheep. Sixty Rambouillet ewes of similar body weight were randomly allocated among three nutritional treatments that were fed during the breeding period (34 days): Control (Control; n = 20), Opuntia (Opuntia; n = 20) and protein-enriched Opuntia (E-Opuntia; n = 20). There were no effects of treatment on body weight but assessment of urine samples indicated that, for 76 metabolites, the Control and Opuntia groups differed completely (P < 0.05), whereas there was overlap between the Control and E-Opuntia groups. It appears that, in Opuntia-fed and Control-fed sheep, different functional groups are activated leading to changes in the metabolism of glucose, tyrosine, methane, and glycerolipids. Fertility and reproductive rate tended to be higher in the Opuntia (70% and 95%) and E-Opuntia (90% and 110%) groups than in the Control (55% and 65%), and an orthogonal contrast revealed the difference between Control and Opuntia to be significant for both reproductive variables (P < 0.05). We conclude that nutritional supplementation with Opuntia cladodes, with or without protein enrichment, increased fertility rate and reproductive rate of female sheep, without any accompanying increases in body weight. Our observations suggest that the reproductive responses to Opuntia do not simply reflect a response to good nutrition, but might be caused by specific metabolites/metabolomic pathways, perhaps by an activation of the metabolism of glucose, methane, tyrosine and glycerolipids. There are few reports relating these metabolomic compounds with the metabolism of the sheep, let alone with reproductive efficiency. The novelty of these discoveries suggests that we need further research into the mechanisms through which nutrition affects the reproductive system.

The use of amino acids during pregnancy, such as glutamine (Gln), seems to be a promising strategy in selected swine breeds to improve the offspring prenatal development. The main goal of the current study was to assess the development of the offspring from parity 1-3 sows of a traditional breed, which were supplemented with 1% glutamine after Day 35 of gestation, under farm conditions. A total of 486 (288 treated) piglets from 78 (46 treated) Iberian sows were used. At birth and slaughterhouse, fatty acid composition, metabolism, and mTOR pathway gene expression were analyzed. At birth, treated newborns showed greater amounts of specific amino acids in plasma, such as glutamine, asparagine, or alanine, and Σn-3 fatty acids in cellular membranes than control newborns. The expression of genes belonging to mTOR Complex 1 was also higher in treated piglets with normal birth-weight. However, these findings did not improve productive traits at birth or following periods in litters from supplemented gilts (parity 1) or sows (parities 2-3). Thus, further research is needed to properly understand the effects of prenatal glutamine supplementation, particularly in traditional swine breeds.

The COVID-19 pandemic, caused by the emergence of a new strain of coronavirus (SARS-CoV-2) around the end of December 2019, has caused a worldwide public health emergency and a socioeconomic crisis during 2020. The lockdown imposed to cope with the health issues caused by the outbreak of the disease has dramatically challenged and negatively affected all the economic sectors of the modern global economy. Specifically, the livestock sector and its related industries are among the most impacted sectors. This is mainly ascribed to the limitations of animal movement and the decrease of production inputs' availability. Other factors negatively affecting the sustainability of the livestock sector have been the shortage of workers due to the lockdown/curfew, the strong decrease in the purchasing power of the consumer, and the intensification of health care tasks. Such an impact is not only highly relevant because of their economic consequences, but also because of the effects of the lockdown and sanitary rules on animal care and welfare. The current review aims to offer: (a) a comprehensive overview of the impact of COVID-19 on the welfare of farm animals and on the performance of livestock farming systems, on food chain sustainability, and finally, on the global economy and food security; and (b) a prospective outline of alleviation actions., (Copyright © 2020 Hashem, González-Bulnes and Rodriguez-Morales.)

Twin-bearing pregnancies of sheep reared in harsh environmental conditions result in maternal undernutrition and feto-maternal oxidative stress, leading to intrauterine growth restriction (IUGR). We assessed the efficiency of supplementation with antioxidant herbal vitamins C and E alone or in combination with concentrate throughout gestation on pregnancy outcomes, pre-weaning growth, and survival of twin lambs from grazing ewes at the Magellan Steppe. Four groups ( n = 30 each) of twin-bearing ewes received a base natural prairie (P) diet, supplemented with either herbal vitamins C 500 mg and E 350 IU per day (V) or concentrated food (S); groups were: P, P + V, P + S, and P + VS. Vitamins and concentrate were supplemented until parturition. At birth, lambs were weighed, and blood was drawn for total antioxidant capacity (TAC) evaluation. Lamb body weight (BW) and survival rate were evaluated at mid-lactation (60 days) and at weaning (120 days). Vitamin supplementation resulted in increased lamb birth weight and TAC, with a trend towards higher BW at weaning, while nutritional supplementation only had a positive effect on birth weight. Lamb survival was higher in both vitamin supplemented groups. In conclusion, supplementation with herbal vitamins C and E alone or in combination with concentrate food during pregnancy may constitute a good nutritional strategy for sheep reared in harsh environmental conditions., Competing Interests: The authors declare no conflicts of interest.

Background: The pathogenesis of renal disease in the context of overweight/obesity, metabolic syndrome, and insulin resistance is not completely understood. This may be due to the lack of a definitive animal model of disease, which limits our understanding of obesity-induced renal damage. We evaluated the changes in renal histology and lipid deposits induced by obesity in a model of insulin resistance: the Iberian swine fed with fat-enriched food., Methods: Twenty-eight female sows were randomized to standard (SD) or high-fat diet (HFD: 6.8% of saturated fat) for 100 days. Weight, adiposity, analytics, oral glucose tolerance tests, and measured renal function were determined. Renal histology and lipid deposits in renal tissue were analyzed., Results: Animals on HFD developed obesity, hypertension, high levels of LDL cholesterol, triglycerides, insulin resistance, and glomerular hyperfiltration. No animal developed overt diabetes. Animals on HFD showed "diabetoid changes", including mesangial expansion [21.40% ± 4 vs.13.20% ± 4.0, p < 0.0001], nodular glomerulosclerosis [7.40% ± 7, 0.75 vs. 2.40% ± 4.7, p = 0.02], and glomerulomegaly (18% vs. 10%, p = 0.010) than those on SD. Tubular atrophy, interstitial fibrosis, inflammation, arteriolar hyalinosis, or fibrointimal thickening were mild and similar between groups. Triglyceride content in renal tissue was higher in animals on HFD than in SD (15.4% ± 0.5 vs. 12.7% ± 0.7; p < 0.01)., Conclusions: Iberian pigs fed with fat-enriched food showed diabetoid changes and glomerulomegaly as observed in obese humans making this model suitable to study obesity-induced renal disease.

Growth is dependent on genotype and diet, even at early developmental stages. In this study, we investigated the effects of genotype, sex, and body weight on the fetal muscle transcriptome of purebred Iberian and crossbred Iberian x Large White pigs sharing the same uterine environment. RNA sequencing was performed on 16 purebred and crossbred fetuses with high body weight (340±14g and 415±14g, respectively) and 16 with low body weight (246±14g and 311±14g, respectively), on gestational day 77. Genotype had the greatest effect on gene expression, with 645 genes identified as differentially expressed (DE) between purebred and crossbred animals. Functional analysis showed differential regulation of pathways involved in energy and lipid metabolism, muscle development, and tissue disorders. In purebred animals, fetal body weight was associated with 35 DE genes involved in development, lipid metabolism and adipogenesis. In crossbred animals, fetal body weight was associated with 60 DE genes involved in muscle development, viability, and immunity. Interestingly, the results suggested an interaction genotype*weight for some DE genes. Fetal sex had only a modest effect on gene expression. This study allowed the identification of genes, metabolic pathways, biological functions and regulators related to fetal genotype, weight and sex, in animals sharing the same uterine environment. Our findings contribute to a better understanding of the molecular events that influence prenatal muscle development and highlight the complex interactions affecting transcriptional regulation during development., Competing Interests: Antonio González-Bulnes and Cristina Ovilo are PLOS One Editorial Board members, however, this does not alter the authors’ adherence to all PLOS One policies.

Intrauterine growth restriction (IUGR) is characterized by reduced growth and weight of the foetus, mainly due to the lack of nutrients and oxygen. Animals affected by IUGR show changes in specific brain areas and several neuronal processes. Female offspring affected by IUGR show increased survival and development compared to males. The objective of this study was to analyse changes in the hippocampus proteome in male and female piglets affected by IUGR. Seven pregnant Iberian sows were fed from Day 35 of pregnancy onwards at 50% of their requirements. At Day 100 of pregnancy, foetuses were obtained and classified by sex and weight, as mild IUGR (Normal Body Weight) versus severe IUGR (Low Body Weight). Hippocampi were dissected and the proteomes analysed by SWATH-MS DIA. In this study, 1497 proteins were identified of which 260 were quantitatively analysed. All differential proteins were more abundant in females versus males and were involved in protein synthesis, neuronal development, metabolism, antiapoptotic signalling and vesicular transport. Our findings support that female foetuses tolerate nutrient limitation better than males, especially under mild IUGR. Under severe IUGR, females still seems to maintain normal lipid metabolism and antiapoptotic signalling, which may be related to the increased female survival. SIGNIFICANCE: In the last years, proteomics have been used to evidence differences related to sex in non-reproductive organs. Intrauterine Growth Restriction (IUGR) can affect female and male offspring differently. Female offspring has stronger protective strategies compared to males, enhancing growth and postnatal survival. Most studies regarding this issue have focused on metabolic organs (i.e. liver). However, the predominance of neurodevelopmental disorders in males suggests that the central nervous system in female offspring adapt better to nutritional stress conditions than that of males. Based on the differential protein expression in hippocampal samples, our work demonstrates that female foetuses indeed adapt better to IUGR than males, especially under mild IUGR conditions. In severe IUGR conditions, differences between males and females were not so evident, but even in this case, the remaining differences suggest increased survival in females than in males., (Copyright © 2019. Published by Elsevier B.V.)

Pregnancy and lactation, especially when concurrent, create a rather metabolically demanding situation in dairy ruminants, but little is known about their effects on offspring phenotype and milk yield. Here, we evaluated the impact of pregnancy and lactation on the metabolic traits and productive performance of Lacaune dairy sheep and their offspring. Productive performance was measured in terms of milk yield, body weight (BW), body condition score (BCS), and size. Productivity was assessed during mid-pregnancy (75 ± 5 d) and late pregnancy (142 ± 4 d) and at 52 ± 5 d in the postpartum period. During pregnancy, high-yielding ewes had higher BW, BCS, plasma glucose, cholesterol, β-OHB, and NEFA than low-yielding ewes, but lower levels of lactate and urea. High-yielding animals had lower BCS after lambing, but their lambs showed greater growth. Productivity during lactation was affected by ewe age and parity: Mature ewes (but not maiden sheep) whose BCS increased steeply during pregnancy yielded more milk in the subsequent lactation than those whose BCS did not increase. Lamb BW and size were positively associated with milk yield in the subsequent lactation. Mature ewes had higher yields than maiden sheep, and mature ewes with multiple pregnancies produced more milk than those with singleton pregnancies. Ewes with male singleton pregnancies also showed higher yield than those with female singletons. These results demonstrate that high-yielding dairy sheep, when appropriately fed and managed, can adequately cover the metabolic demands of pregnancy and high milk production (even when concurrent) without losing productivity.

Pregnancy and lactation are challenging states that affect maternal and lamb health. In Lacaune dairy sheep, we evaluated the impact of parity, pregnancy rank, and body condition on body weight and the condition of ewes and lambs in mid-pregnancy (75 ± 5 d), in late pregnancy (142 ± 4d), and postpartum (52 ± 5d pp). Maternal age was associated with initial decreases, followed by increases, in body weight and condition. After lambing, both mature and maiden ewes lost weight and body condition. Maternal indices of glucose, protein, and lipid metabolism were within physiological values during pregnancy, but postpartum values depended on maternal parity and pregnancy rank, with multiple-pregnant ewes showing a postpartum increase in glucose and maiden sheep a postpartum increase in plasma cholesterol concentration. Male lambs were heavier than female lambs at birth, and lambs born to mothers with higher body condition scores were heavier. Lambs born as singletons were heavier than those born in litters. Maternal age and pregnancy rank did not influence lamb metabolic indicators. Sex affected plasma concentrations of glucose, triglycerides, and cholesterol. Maternal metabolic indicators showed minimal effects on lamb phenotype. These results suggest that, when appropriately fed, dairy sheep can cover the metabolic demands of pregnancy and milk production, regardless of age and pregnancy rank.

Twinning and maternal nutritional restriction leads to fetal hypoxia, oxidative stress, and intrauterine growth restriction (IUGR) in near-term sheep pregnancies. Our aim was to determine the effect of oral supplementation of vitamins C and E in pregnant sheep on maternal and umbilical cord blood concentrations of vitamins C and E and the effects on fetal antioxidant status, growth, and placental efficiency. Sixteen single- and sixteen twin-bearing ewes, grazing natural Patagonian prairies, were selected after transrectal ultrasound at day 30 after mating. Half of ewes from each pregnancy rank were supplemented daily with vitamins C and E, administered orally, from 30 to 140 days of gestation, when maternal jugular and fetal venous cord blood samples were obtained during cesarean section. Fetuses were weighed and sexed. Placental weight in each fetus was also obtained. Blood plasma was harvested for measurements of maternal and fetal vitamins concentration and fetal antioxidant capacity. Maternal administration of vitamin C and E was associated with increased fetal cord levels of both vitamins, improved antioxidant status, and enhanced fetal growth in both singleton and twin pregnancies associated with increased placental efficiency. These results highlight the potential of vitamin C and E supplementation to reduce the impact of IUGR in both livestock and humans.

Fetal hypoxia, resulting in oxidative stress in pregnancies, contributes to reduced fetal growth. Melatonin, a potent antioxidant, has been associated with improved oxidative status. Maternal oral melatonin supplementation in sheep from day 50 of gestation ameliorates the consequences of fetal growth restriction in sheep. In rats, melatonin supplementation increases fetal weight via improved placental efficiency and reduction of oxidative stress. The objective of this study was to evaluate whether melatonin supplementation of single (S)- or twin-bearing (T) ewes using either 0 (0MEL), 1 (18-mg MEL), or 2 (36-mg MEL) slow release 18-mg melatonin implants (Regulin) from 100 to 140 d of pregnancy (n = 8 per group) influenced fetal oxygen supply and fetal weight. Fetal umbilical vein blood samples were collected at P140 and partial pressure of oxygen (PO2) and hemoglobin saturation by oxygen (SatHb) measured. The placenta from each fetus was excised and placentomes individually weighed and typed (A-D). Pregnancy rank, sex of the fetus, number of implants, and their interaction on fetal weight, blood gases, and placentome weight were analyzed using ANOVA. A 22% and 14% increase (P < 0.05) in body weight was obtained in 36- and 18-mg MEL compared with 0 MEL twin male fetuses, respectively, but no treatment effects were observed in singletons or females from twin pregnancies. Fetuses from ewes receiving 36-mg MEL had an 18% to 20% increase in cord PO2 (P < 0.05) compared with 18-mg MEL and 0MEL fetuses, which in turn did not differ. Fetal weight was positively correlated with PO2 (r = 0.37; P = 0.02), SatHb (r = 0.26; P = 0.03), and O2 content (r = 0.236; P = 0.048). No treatment effect on placentome average weight, total placentome weight per fetus or per ewe, nor total number of placentomes per fetus was observed. However, placentae from 36-mg MEL fetuses had a greater proportion of Type C (P < 0.05) than 0MEL and 18-mg MEL ewes, and tended to have a lower proportion of Type A (P = 0.1) and a greater proportion of Type D (P = 0.06) placentomes, compared with 0MEL ewes. These results indicate that maternal melatonin implants, independently of sex, improve oxygen supply to the fetus, which could potentially improve lamb vigor at birth. In addition, melatonin can increase fetal weight of twin males, by improving placental adaptation and fetal blood oxygenation.

Low birth weight has profound implications for perinatal mortality and morbidity in lambs, causing higher mortality and lower growth potential. Low birth weight, as a consequence of fetal growth restriction, occurs in undernourished and multiple pregnancies, where hypoxia and oxidative stress could play a critical role. Our aim was to establish the effects of nutritional deprivation and pregnancy rank on fetal growth, oxygenation, and oxidative status in sheep pregnancies under extensive Patagonian conditions. At 30 days after mating, single- and twin-bearing ewes were offered only natural pasture (undernutrition group) or natural pasture plus concentrate supplementation (well-nourished group). At day 140 of gestation, blood gases and redox status were evaluated in venous cord blood, and fetal biometric characteristics were obtained after cesarean section. Both maternal undernutrition and twinning led to decreased oxygen supply to the fetuses ( p = 0.016 and p = 0.050, respectively), which was associated with decreased intrauterine growth (r = 0.446, p < 0.01). Moreover, twinning increased oxidative stress in cord blood ( p < 0.05), which might also contribute to fetal growth restriction. These results reinforce the importance of maternal nutrition, especially for those ewes bearing multiples, and opens new possibilities for nutritional or antioxidant interventions for preventing fetal hypoxia and oxidative stress.

There is no simple method to measure glomerular filtration rate (GFR) in swine, an established model for studying renal disease. We developed a protocol to measure GFR in conscious swine by using the plasma clearance of iohexol. We used two groups, test and validation, with eight animals each. Ten milliliters of iohexol (6.47 g) was injected into the marginal auricular vein and blood samples (3 mL) were collected from the orbital sinus at different points after injection. GFR was determined using two models: two-compartment (CL2: all samples) and one-compartment (CL1: the last six samples). In the test group, CL1 overestimated CL2 by ~30%: CL2 = 245 ± 93 and CL1 = 308 ± 123 mL/min. This error was corrected by a first-order polynomial quadratic equation to CL1, which was considered the simplified method: SM = -47.909 + (1.176xCL1) - (0.00063968xCL1²). The SM showed narrow limits of agreement with CL2, a concordance correlation of 0.97, and a total deviation index of 14.73%. Similar results were obtained for the validation group. This protocol is reliable, reproducible, can be performed in conscious animals, uses a single dose of the marker, and requires a reduced number of samples, and avoids urine collection. Finally, it presents a significant improvement in animal welfare conditions and handling necessities in experimental trials., Competing Interests: The authors declare no conflict of interest.

The present study evaluated the effectiveness of sildenafil citrate (SC) to improve placental and fetal growth in a diet-induced rabbit model of intrauterine growth restriction (IUGR). Pregnant rabbits were fed either ad libitum (Group C) or restricted to 50% of dietary requirements (Group R) or restricted and treated with SC (Group SC). The treatment with SC improved placental development by increasing vascularity and vessel hypertrophy in the decidua. The assessment of feto-placental haemodynamics showed higher resistance and pulsatility indices at the middle cerebral artery (MCA) in fetuses treated with SC when compared with Group R, which had increased systolic peak and time-averaged mean velocities at the MCA. Furthermore, fetuses in the SC group had significantly higher biparietal and thoracic diameters and longer crown-rump lengths than fetuses in Group R. Hence, the SC group had a reduced IUGR rate and a higher kit size at birth compared with Group R. In conclusion, SC may provide potential benefits in pregnancies with placental insufficiency and IUGR, partially counteracting the negative effects of food restriction on placental development and fetal growth. However, the present study also found evidence of a possible blood overflow in the brain that warrants further investigation.

Iberian pig production includes purebred (IB) and Duroc-crossbred (IBxDU) pigs, which show important differences in growth, fattening and tissue composition. This experiment was conducted to investigate the effects of genetic type and muscle (Longissimus dorsi (LD) vs Biceps femoris (BF)) on gene expression and transcriptional regulation at two developmental stages. Nine IB and 10 IBxDU piglets were slaughtered at birth, and seven IB and 10 IBxDU at four months of age (growing period). Carcass traits and LD intramuscular fat (IMF) content were measured. Muscle transcriptome was analyzed on LD samples with RNA-Seq technology. Carcasses were smaller in IB than in IBxDU neonates (p < 0.001), while growing IB pigs showed greater IMF content (p < 0.05). Gene expression was affected (p < 0.01 and Fold change > 1.5) by the developmental stage (5,812 genes), muscle type (135 genes), and genetic type (261 genes at birth and 113 at growth). Newborns transcriptome reflected a highly proliferative developmental stage, while older pigs showed upregulation of catabolic and muscle functioning processes. Regarding the genetic type effect, IBxDU newborns showed enrichment of gene pathways involved in muscle growth, in agreement with the higher prenatal growth observed in these pigs. However, IB growing pigs showed enrichment of pathways involved in protein deposition and cellular growth, supporting the compensatory gain experienced by IB pigs during this period. Moreover, newborn and growing IB pigs showed more active glucose and lipid metabolism than IBxDU pigs. Moreover, LD muscle seems to have more active muscular and cell growth, while BF points towards lipid metabolism and fat deposition. Several regulators controlling transcriptome changes in both genotypes were identified across muscles and ages (SIM1, PVALB, MEFs, TCF7L2 or FOXO1), being strong candidate genes to drive expression and thus, phenotypic differences between IB and IBxDU pigs. Many of the identified regulators were known to be involved in muscle and adipose tissues development, but others not previously associated with pig muscle growth were also identified, as PVALB, KLF1 or IRF2. The present study discloses potential molecular mechanisms underlying phenotypic differences observed between IB and IBxDU pigs and highlights candidate genes implicated in these molecular mechanisms., Competing Interests: The authors have declared that no competing interests exist, excepting that the coauthors Antonio Gonzalez-Bulnes and Cristina Óvilo are PLOS ONE Editorial Board members. However, this does not alter the authors’ adherence to all the PLOS ONE policies.

Iberian ham production includes both purebred (IB) and Duroc-crossbred (IBxDU) Iberian pigs, which show important differences in meat quality and production traits, such as muscle growth and fatness. This experiment was conducted to investigate gene expression differences, transcriptional regulation and genetic polymorphisms that could be associated with the observed phenotypic differences between IB and IBxDU pigs. Nine IB and 10 IBxDU pigs were slaughtered at birth. Morphometric measures and blood samples were obtained and samples from Biceps femoris muscle were employed for compositional and transcriptome analysis by RNA-Seq technology. Phenotypic differences were evident at this early age, including greater body size and weight in IBxDU and greater Biceps femoris intramuscular fat and plasma cholesterol content in IB newborns. We detected 149 differentially expressed genes between IB and IBxDU neonates (p < 0.01 and Fold-Change > 1. 5). Several were related to adipose and muscle tissues development (DLK1, FGF21 or UBC). The functional interpretation of the transcriptomic differences revealed enrichment of functions and pathways related to lipid metabolism in IB and to cellular and muscle growth in IBxDU pigs. Protein catabolism, cholesterol biosynthesis and immune system were functions enriched in both genotypes. We identified transcription factors potentially affecting the observed gene expression differences. Some of them have known functions on adipogenesis (CEBPA, EGRs), lipid metabolism (PPARGC1B) and myogenesis (FOXOs, MEF2D, MYOD1), which suggest a key role in the meat quality differences existing between IB and IBxDU hams. We also identified several polymorphisms showing differential segregation between IB and IBxDU pigs. Among them, non-synonymous variants were detected in several transcription factors as PPARGC1B and TRIM63 genes, which could be associated to altered gene function. Taken together, these results provide information about candidate genes, metabolic pathways and genetic polymorphisms potentially involved in phenotypic differences between IB and IBxDU pigs associated to meat quality and production traits.

Background: During freezing the selective precipitation of substances in the medium may provoke a pH shift and lead to sperm damage., Objective: To study the effect of the pH pre-adjustment in the freezing extender on post-thaw boar sperm quality., Methods: A total of 15 ejaculates from different boars were obtained and divided into six aliquots prior to a standard straw cryopreservation in freezing extender (lactose-egg yolk-glycerol-Orvus ES Paste) with different pH. After thawing, sperm quality (plasma membrane integrity, motility and acrosome status) were assessed at 30 and 90 minutes of post-thaw incubation at 37 degree C., Results: When the boar sperm were frozen in a freezing media with pH basic, and particularly at pH 8, it had higher post-thaw sperm quality., Conclusion: The pre-adjustment at pH 8 of the freezing extender (lactose-egg yolk-glycerol-Orvus ES Paste) is able to improve the post-thaw boar sperm quality.

Maternal energy restriction during pregnancy predisposes to metabolic alterations in the offspring. The present study was designed to evaluate phenotypic and metabolic consequences following maternal undernutrition in an obese pig model and to define the potential role of hypothalamic gene expression in programming effects. Iberian sows were fed a control or a 50 % restricted diet for the last two-thirds of gestation. Newborns were assessed for body and organ weights, hormonal and metabolic status, and hypothalamic expression of genes implicated in energy homeostasis, glucocorticoid function and methylation. Weight and adiposity were measured in adult littermates. Newborns of the restricted sows were lighter (P <0·01), but brain growth was spared. The plasma concentration of TAG was lower in the restricted newborns than in the control newborns of both the sexes (P <0·01), while the concentration of cortisol was higher in females born to the restricted sows (P <0·04), reflecting a situation of metabolic stress by nutrient insufficiency. A lower hypothalamic expression of anorexigenic peptides (LEPR and POMC, P <0·01 and P <0·04, respectively) was observed in females born to the restricted sows, but no effect was observed in the males. The expression of HSD11B1 gene was down-regulated in the restricted animals (P <0·05), suggesting an adaptive mechanism for reducing the harmful effects of elevated concentrations of cortisol. At 4 and 7 months of age, the restricted females were heavier and fatter than the controls (P< 0·01). Maternal feed restriction induces asymmetrical growth retardation and metabolic alterations in the offspring. Differences in gene expression at birth and higher growth and adiposity in adulthood suggest a female-specific programming effect for a positive energy balance, possibly due to overexposure to endogenous stress-induced glucocorticoids.

Background: At high altitudes, hypoxia, oxidative stress or both compromise sheep fertility. In the present work, we tested the relative effect of short- or long-term exposure to high altitude hypobaric hypoxia and oxidative stress on corpora luteal structure and function., Methods: The growth dynamics of the corpora lutea during the estrous cycle were studied daily by ultrasonography in cycling sheep that were either native or naïve to high-altitude conditions and that were supplemented or not supplemented with antioxidant vitamins. Arterial and venous blood samples were simultaneously drawn for determination of gases and oxidative stress biomarkers and progesterone measurement. On day five after ovulation in the next cycle, the ovaries were removed for immunodetection of luteal HIF-1alpha and VEGF and IGF-I and to detect IGF-II gene expression., Results: The results showed that both short- and long-term exposure to high-altitude conditions decreased luteal growth and IGF-I and IGF-II gene expression but increased HIF-1 alpha and VEGF immunoexpression. The level of plasma progesterone was also increased at a high altitude, although an association with increased corpus luteum vascularization was only found in sheep native to a high-altitude location. Administration of antioxidant vitamins resulted in a limited effect, which was restricted to decreased expression of oxidative stress biomarkers and luteal HIF-1alpha and VEGF immunoexpression., Conclusions: Exposure of the sheep to high-altitude hypobaric hypoxia for short or long time periods affects the development and function of the corpus luteum. Moreover, the observed association of oxidative stress with hypoxia and the absence of any significant effect of antioxidant vitamins on most anatomical and functional corpus luteum traits suggests that the effects of high altitude on this ovarian structure are mainly mediated by hypoxia. Thus, these findings may help explain the decrease in sheep fertility at a high altitude.

Sheep pregnancy in high-altitude environments frequently involves hypoxia and oxidative stress and causes intrauterine growth retardation. The adverse effects of altitude on fetal growth can be prevented by the administration of antioxidant vitamins, but the mechanisms responsible are not well known. The maintenance of a viable pregnancy depends largely on adequate placental steroidogenesis, especially in the last two-thirds of pregnancy. Thus, in the present study we evaluated the effect of antioxidant vitamins (C and E) on plasma concentrations of progesterone and 17β-oestradiol during the last two-thirds of high-altitude pregnancies in ewes both native and naïve to the high-altitude environment. In addition, pregnancy outcomes were evaluated by determining the bodyweight of newborn lambs. Sex steroid patterns differed between ewes with and without vitamin supplementation. Concentrations of plasma progesterone and 17β-oestradiol were significantly higher in the supplemented groups from approximately 40 days before parturition until near term. Newborn weights were significantly lower in animals not adapted to the higher altitude, and vitamin supplementation prevented this decrease. In conclusion, the administration of antioxidant vitamins in the present study enhanced placental steroidogenesis, thus favouring fetal development in pregnancies developing at high altitudes.

The aim of the present study was to evaluate the inclusion of seminal plasma (SP) in the freezing extender, trying to preserve as much as possible of SP with spermatozoa from Iberian pigs, thus improving the conservation of animal genetic resources of this breed. Experiment 1, evaluated the effect of substituting water with SP as diluent in the freezing media in different proportions (0%, 10%, 25%, 50%, 75% and 100%), over pre-freezing (at 10°C and 5°C) and post-thawing sperm quality. The results showed that over 50% of SP in the extender, significantly decreased sperm quality in comparison to the control sample (0% SP) and the samples with 10% and 25% of SP (P<0.05). No significant differences were found between the control sample and the samples with 10% and 25% SP (P>0.05), but treatment with 25% did not show significant differences between the time of incubation at 37°C after thawing (P>0.05), showing greater sperm quality resistance over time. Experiment 2, evaluated the effect of prolonged incubation period, until 480min (simulating the lifespan of sperm in the female genital tract), of sperm samples with 0%, 10% and 25% of SP. Treatment with 25% of SP maintained better sperm quality over time, compared to control sample. Significant differences were observed especially in the parameters of motility analysis (TMS, total motile spermatozoa; PMS: progressive motility spermatozoa. P<0.05). In Experiment 3, the effect of the presence of SP was evaluated during the thawing process. Although some differences were observed between treatments, these differences were not as clear as the previous experiments. In conclusion, replacement of 25% of the water by SP as diluent in the freezing extender could be considered the maximum percentage of inclusion, without harmful effects to the sperm. In addition, this proportion of SP maintained Iberian sperm quality for longer time when it was present during the freezing and thawing process., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The effect of betacarotene (BC) supplementation on the onset of puberty and serum insulin levels in goats was evaluated in the study. In June, prepuberal goats (n=17; 3 months old; 7/8 Saanen-Alpine; 26° NL) were randomly assigned to one of two groups: 1/ betacarotene group supplemented daily with 50 mg of BC (n=9; live weight [LW]: 17.3±1.0 kg; body condition score [BCS]: 3.34±0.12) or 2/ control group (CONT; n=8; LW:16.1±1.0 kg; BCS=3.17±0.12). From June to November, an intermittent blood sampling was performed twice per week in both groups to evaluate serum progesterone (P(4)), while monthly samples were intended for insulin (INS) determination. Initial mean LW (16.7±1.0 kg) and BCS (3.31±0.12) were similar (p>0.05) in both groups. Mean serum insulin (1.37 vs. 1.18±0.09 ng/ml), age of puberty (215.7 vs. 226.5±6.6 days) and the percentage of goats reaching puberty (44.4 vs. 25.0±17.0%) did not differ (p>0.05) between BC and CONT group, respectively. However, increase in serum insulin during the second half of the experiment was observed in BC group (p<0.05) which was positively correlated with LW (r=0.95; p<0.05). In addition, as LW (r=-0.89) and serum insulin (r=-0.76) levels increased, the natural photoperiod decreased, revealing negative correlations (p<0.05) between the respective variables. In this study, BC supplementation did not promote precocious puberty and did not affect the percentage of goats reaching activation of the hypothalamic-hypophyseal-gonadal axis during the establishment of puberty. Nonetheless, BC supplementation positively affected the release pattern of insulin suggesting a potential role of BC as pancreas-activating molecule.

Small ruminant species such as sheep and goats are short-day breeders, which is a crucial factor affecting the offer of lambs and kids throughout the year. An appropriate management of reproduction allows ewes and does to breed in the spring to increase the supply of product to the marketplace on a year-round basis. Pharmaceutical control of reproduction is possible, usually through administration of hormones or analogues related to the natural estrous cycle, such as progesterone, prostaglandins, and/or melatonin., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Seasonal changes in ovulatory activity, assessed by the measurement of plasma progesterone and plasma prolactin concentrations were monitored in 10 Spanish ibex females captured in the National Wildlife Reserve of Sierra Nevada (South Spain, 37 degrees N). Five of the 10 female ibex showed ovulatory activity with a mean (+/- s.e.m.) duration of the oestrous cycle of 19.4 +/- 1 days (range: 17-23 days). On average, the five cyclic females weighed 28 +/- 0.9 kg. Progesterone cycles occurred only in animals older than 4 years of age. Ovulatory activity extended from December to January. The duration of the breeding season was 43.2 +/- 7.7 days. Ibex females younger than 4 years of age had a body weight lower (P < 0.01 ) than that of adults and none of them displayed ovulations. Plasma concentrations of prolactin levels were significantly affected by season (P < 0.05), following a trend that was roughly parallel to daylength. The highest values occurred in the spring (119.7 +/- 21.4 ng x mL(-1)) and the summer (139.3 +/- 19.8 ng x mL(-1)), the lowest values in the autumn (26 +/- 7.4 ng x mL(-1)) and in the winter (19.7 +/- 3.2 ng x mL(-1)). Our results showed a very restricted breeding season, despite the fact that the Spanish ibex originates from and lives in a temperate latitude, revealing a remarkably good adaptation to the harsh climatic and nutritional conditions of their mountainous habitat.

In a first experiment, embryo viability was estimated after recovery in the uterus or the oviduct of 70 Manchega ewes following a treatment of superovulation with decreasing doses of OVAGEN. Fewer viable embryos (5.6 +/- 0.9 vs. 8.3 +/- 0.8, P < 0.05) and more degenerative embryos (31.3% vs. 6.8%, P < 0.005) were obtained from the uterus than from the oviduct respectively. In a second experiment performed on 14 ewes, embryo viability was analyzed in relation to the follicular population estimated by ultrasonography (follicles > or = 2 mm) at the first FSH administration. Progesterone (P4) and oestradiol 17beta (E2) concentrations were also determined from the beginning of the superovulation treatment to the recovery of the embryos. The number of viable embryos (4.3 +/- 1.4) was positively correlated (r = 0.824) with of 2-4 mm diameter follicles (P < 0.05), and with E2 concentrations at -12 h (r = 0.891, P < 0.01) , 0 h (r = 0.943, P < 0.0001) and +24 h (r = 0.948, P < 0.05) from estrus detection. Prolonged high levels of E2 up to 72 h with low levels of P4 on days 3 and 4 after estrus had a negative (P < 0.05) effect on embryo viability. These results indicate that ovarian response to superovulatory protocols is related to the individual variations in the number of follicles of 2-4 mm at the start of FSH treatment, and that embryo viability is conditioned by the steroid patterns during the time spent in the genital tract of the super-ovulated ewes.