[EN] The aim of this study was to distinguish effects due to diet composition from those triggered by ruminal pH on fermentation patterns and microbial profiles in a continuous culture system (RUSITEC). The study followed a 2 × 2 factorial design, with two diets varying in the proportions of forage and concentrate and two pH levels in the culture medium. RUSITEC fermenters were used to simulate rumen fermentation and feed digestibility, fermentation end-products, microbial protein synthesis, microbial community, and long-chain fatty acid profiles in the digesta were determined. Multivariate analyses were applied to summarize the overall results. High concentrate (34% cereal grain, 32% hay) diets were more digestible (p < 0.05) than high forage (10% cereal grain, 78% hay) diets, resulting in a greater (p < 0.05) formation of most fermentation end-products and microbial protein in the rumen. However, there were no significant (p > 0.05) differences between diets in methane production. Ciliate protozoa, anaerobic fungi, some fibrolytic bacteria, hydrogenation of oleic acid, and relative proportion of conjugated linoleic acid were increased (p < 0.05) with high forage diets. A decline in rumen pH from 6.8 to 6.4 decreased (p < 0.05) feed digestibility, protein degradability, and the daily outputs of some fermentation end-products (gas, VFA, acetate, ammonia) but had no effect (p > 0.05) on the synthesis of microbial protein, and on the output of methane, propionate, butyrate or lactate. Minor changes in microbial community profile or the fatty acid relative proportions were observed within this pH range. The overall multivariate analysis revealed a clear discrimination between high-concentrate and high-forage diets, with subtler and less-defined pH effects on ruminal fermentation and microbial communities, SI, This work was funded by the Spanish Ministerio de Ciencia e Innovación (PID2021- 126489OB-I00, MCIN/AEI/10.13039/501100011033) and co-funded by the European Regional Development Fund (ERDF-FEDER) ”A way to make Europe-Una manera de hacer Europa”). Funding for the payment of APC was provided by Universidad de León (Ayudas a Grupos de Investigación BB202)

[EN]Adequate management of replacement ewe lambs (F0) in dairy sheep farms during postnatal life may modify the germline cells, thus promoting transmission of intergenerational effects to the offspring (F1). To test this hypothesis, 18 newborn male lambs (F1), either born from methionine-supplemented ewe lambs (F0 ewe lambs being fed ad libitum with a milk replacer supplemented with 1 g methionine/kg DM) or not supplemented (F0 ewe lambs being fed ad libitum with the same milk replacer with no methionine added), were included in the present study. All the male F1 lambs were managed exactly in the same way along the whole lifespan in order to bring out the differences caused by methionine supplementation of F0 dams. Our data show that the methionine supplementation of dams (F0) during the suckling period did not promote significant (p > 0.05) changes on feed intake, growth rate, or feed efficiency of F1 male lambs during the fattening period. Moreover, the meat chemical composition (proximal, fatty acid profile, and volatile compounds) was similar for both groups (p > 0.05), but the meat of F1-MET lambs presented higher redness and hardness (p < 0.05) when compared to F1-CTRL lambs. The biochemical profile also highlighted significant (p < 0.05) differences in the serum creatinine and calcium content that may be at least partially related to the meat quality traits observed. Overall, all these results suggest that methionine supplementation of lambs (F0) during early postnatal life causes permanent changes in the offspring. This has positive effects, such as achieving a more attractive color of lamb meat (F1) for consumers, and negative effects, such as reduced meat tenderness., SI, This work was funded by Ministerio de Ciencia e Innovacion (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”). Alba Martin gratefully acknowledges receipt of a pre-doctoral grant (PRE2019-089288) from Ministerio de Ciencia e Innovación MCIN/AEI/10.13039/501100011033, ”El FSE invierte en tu futuro”).

[EN] The effects of pelleting a total mixed ration (TMR) for dairy sheep were investigated in an experiment involving 24 lactating Assaf ewes, which were assigned to two groups and fed the same TMR ad libitum, offered either in pelleted (PTMR group, n = 12) or in unpelleted form (CTMR group, n = 12). The experiment lasted 28 days, during which feed intake, eating behavior (including meal frequency and size, meal duration, eating rate, between-meal interval), and milk yield were recorded daily. Body weight (BW) was recorded on days 1 and 28 and milk samples were collected on days 1, 8, 15, 22 and 28 for milk composition analysis. Blood acid-base status was determined at the beginning and at the end of the trial. Ewes fed the CTMR diet exhibited (p < 0.05) a higher meal frequency and longer meal duration, along with a smaller meal size and slower eating rate. However, feed intake in this group was less than that in ewes fed PTMR only during the final two weeks of the experimental period. Total eating time was also longer (p < 0.001) in the CTMR group, whereas the average time between meals was shorter (p < 0.002). No differences (p > 0.05) were observed between dietary treatments in blood acid-base status, milk yield or milk composition. However, a diet x day interaction (p < 0.05) was detected for milk yield, as during the last 2 weeks of the experimental period the ewes fed the PTMR yielded more milk than those fed the CTMR. Feed conversion ratio did not differ between groups (p > 0.05), but body weight loss was greater in ewes fed the CTMR diet (−3.00 vs. −0.58 kg; p < 0.05). A trend toward improved feed efficiency was observed in the PTMR group when calculated based on milk yield corrected for that theoretically derived from the mobilization of body reserves (1.98 vs. 1.41 g DMI/kg milk; p = 0.077), with estimated contributions from body reserves of 485 g/day in the CTMR group and 70 g/day in the PTMR group. In conclusion, the use of pelleted total mixed rations in high-yielding dairy ewes enhances feed intake, feed efficiency, milk yield, and energy balance without adversely affecting milk composition or animal health in the short term., SI, This work was funded by Ministerio de Ciencia e Innovación (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”)., The collaboration of the Animal Selection and Reproduction Center (CENSYRA, Junta de Castilla y León, Spain) in the analysis of milk samples is gratefully acknowledged.

[EN]Methionine supplementation during the suckling period of ewe lambs may modify DNA methylation in the germline (F0) during this critical window period for the neonate. This circumstance may promote the intergenerational transmission of epigenetic marks to the offspring (F1), thus altering the expression of specific genes and physiological traits throughout F1 life. To test this hypothesis, 27 newborn ewe lambs (F1) born from either ewes being supplemented 0.1 % methionine (dry matter basis) during the suckling period (F0-MET) or not supplemented (F0-CTRL) were assigned to two different groups (F1-CTRL, n = 13 and F1-MET, n = 14), being the dietary treatment of their mothers (F0) the only source of variation. Thus, all the F1 animals (both groups) were raised exactly in the same way along the whole life (including lactation). In this study, we determined differences in the global blood methylation patterns, biochemical profile, and metabolome of female offspring (F1). Our data showed that functional categories such as those related to developmental process and anatomical structure development were significantly enriched in the F1-MET ewe lambs due to differentially methylated regions of genes in these categories. These F1-MET ewes also presented lower live body weight (P < 0.05) and reduced lipomobilization and milk yield (P = 0.099) during the lactation period, together with increased PUFAs content (P = 0.075) in the milk fatty acid profile when compared to F1-CTRL. Increased levels of insulin (P = 0.031) and β-hydroxybutyrate (P = 0.043), along with certain features of the metabolome profiles, revealed altered lipid metabolism when compared to F1-CTRL animals. As far as fecal microbiota is concerned, no significant differences were found in alpha and beta diversity between the two groups. However, DESeq2 analysis performed on OTU-filtered data revealed that Treponema (genus) and Spirochaetaceae (family), both from the Spirochaetes phylum, were reduced in the F1-MET group compared to the F1-CTRL group (log2FC=-3.96, padj < 0.05). The results suggest that early methionine supplementation in F0 ewe lambs has an intergenerational impact on their F1 female offspring, with negative consequences on lipomobilization and milk production during F1 lactation., SI, This work was funded by Ministerio de Ciencia e Innovaci´on (PID2021–126489OB-I00, MCIN/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”).

[EN]Nutritional strategies that supply methyl donors, such as methionine in the early post-natal period of lambs could program feed efficiency, milk production and metabolism along the whole life due to persistent changes in epigenetic marks. To test this hypothesis, 34 newborn ewe lambs from the same flock were stratified and distributed into two groups with equal body weight distribution. The control group (F0-CTRL, n = 17) was fed ad libitum with a milk replacer. In contrast, the second group (F0-MET, n = 17) received the same milk replacer supplemented with 1 g methionine/kg DM. After weaning, all animals were raised the same way and fed ad libitum with a complete pelleted diet formulated according to nutrient requirements. At 45 days of age, blood was collected from the jugular vein for DNA isolation, and DNA methylation was analyzed by Reduced Representation Bisulfite Sequencing (RRBS) in ten animals [5 ewe lambs (F0-MET) vs. 5 ewe lambs (F0-CTRL), corresponding to 5 twin births, of which one lamb was assigned to each group]. The main results show that CPT1B, a gene involved in fatty acid catabolism, was differentially methylated in F0-MET lambs (45 days of life). In addition, these animals presented increased insulin and non-esterified fatty acid concentrations while cholesterol levels were reduced, most of these changes being persistent along the whole life. No differences were observed in milk production and feed efficiency during lactation. Still, the milk's fatty acid (FA) profile showed increased levels of long-chain fatty acids, n3 and n6 polyunsaturated fatty acids. All these results suggest that methionine supplementation in early life can modify ewes' milk fatty acid profile without compromising feed efficiency. However, it is necessary to clarify whether this effect is passed on to the offspring (F1) through epigenetic marks in the germline., SI, This work was funded by Ministerio de Ciencia e Innovación (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033, "FEDER, Una manera de hacer Europa"). Alba Martín gratefully acknowledges receipt of a pre-doctoral grant (PRE2019-089288) from Ministerio de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033, "El FSE invierte en tu futuro". The metabolomics analysis was carried out at the UNI-TECH "OMICS" platforms of the Università degli Studi di Milano. We acknowledge Dr. Manuela Fontana’s necessary support from the UNI-TECH "OMICS" platforms of the Università degli Studi di Milano. CENSYRA (León, Spain) is also gratefully acknowledged for milk analyses.

The effects of pelleting a total mixed ration (TMR) for dairy sheep were investigated in an experiment involving 24 lactating Assaf ewes, which were assigned to two groups and fed the same TMR ad libitum, offered either in pelleted (PTMR group, n = 12) or in unpelleted form (CTMR group, n = 12). The experiment lasted 28 days, during which feed intake, eating behavior (including meal frequency and size, meal duration, eating rate, between-meal interval), and milk yield were recorded daily. Body weight (BW) was recorded on days 1 and 28 and milk samples were collected on days 1, 8, 15, 22 and 28 for milk composition analysis. Blood acid-base status was determined at the beginning and at the end of the trial. Ewes fed the CTMR diet exhibited (p < 0.05) a higher meal frequency and longer meal duration, along with a smaller meal size and slower eating rate. However, feed intake in this group was less than that in ewes fed PTMR only during the final two weeks of the experimental period. Total eating time was also longer (p < 0.001) in the CTMR group, whereas the average time between meals was shorter (p < 0.002). No differences (p > 0.05) were observed between dietary treatments in blood acid-base status, milk yield or milk composition. However, a diet x day interaction (p < 0.05) was detected for milk yield, as during the last 2 weeks of the experimental period the ewes fed the PTMR yielded more milk than those fed the CTMR. Feed conversion ratio did not differ between groups (p > 0.05), but body weight loss was greater in ewes fed the CTMR diet (−3.00 vs. −0.58 kg; p < 0.05). A trend toward improved feed efficiency was observed in the PTMR group when calculated based on milk yield corrected for that theoretically derived from the mobilization of body reserves (1.98 vs. 1.41 g DMI/kg milk; p = 0.077), with estimated contributions from body reserves of 485 g/day in the CTMR group and 70 g/day in the PTMR group. In conclusion, the use of pelleted total mixed rations in high-yielding dairy ewes enhances feed intake, feed efficiency, milk yield, and energy balance without adversely affecting milk composition or animal health in the short term., This work was funded by Ministerio de Ciencia e Innovación (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”)., info:eu-repo/semantics/publishedVersion

19 páginas, 4 tablas, 3 figuras., The aim of this study was to distinguish effects due to diet composition from those triggered
by ruminal pH on fermentation patterns and microbial profiles in a continuous culture system
(RUSITEC). The study followed a 2 × 2 factorial design, with two diets varying in the proportions of
forage and concentrate and two pH levels in the culture medium. RUSITEC fermenters were used to
simulate rumen fermentation and feed digestibility, fermentation end-products, microbial protein
synthesis, microbial community, and long-chain fatty acid profiles in the digesta were determined.
Multivariate analyses were applied to summarize the overall results. High concentrate (34% cereal
grain, 32% hay) diets were more digestible (p < 0.05) than high forage (10% cereal grain, 78% hay)
diets, resulting in a greater (p < 0.05) formation of most fermentation end-products and microbial
protein in the rumen. However, there were no significant (p > 0.05) differences between diets in
methane production. Ciliate protozoa, anaerobic fungi, some fibrolytic bacteria, hydrogenation of
oleic acid, and relative proportion of conjugated linoleic acid were increased (p < 0.05) with high
forage diets. A decline in rumen pH from 6.8 to 6.4 decreased (p < 0.05) feed digestibility, protein
degradability, and the daily outputs of some fermentation end-products (gas, VFA, acetate, ammonia)
but had no effect (p > 0.05) on the synthesis of microbial protein, and on the output of methane,
propionate, butyrate or lactate. Minor changes in microbial community profile or the fatty acid
relative proportions were observed within this pH range. The overall multivariate analysis revealed
a clear discrimination between high-concentrate and high-forage diets, with subtler and less-defined
pH effects on ruminal fermentation and microbial communities., This work was funded by the Spanish Ministerio de Ciencia e Innovación (PID2021-
126489OB-I00, MCIN/AEI/10.13039/501100011033) and co-funded by the European Regional Development Fund (ERDF-FEDER) ”A way to make Europe-Una manera de hacer Europa”). Funding
for the payment of APC was provided by Universidad de León (Ayudas a Grupos de Investigación BB202)., Peer reviewed

Methionine supplementation during the suckling period of ewe lambs might program feed efficiency during the whole life time due to stable changes of epigenetic marks caused by methylation of DNA. The results of the present study revealed that methionine supplementation during the suckling period has a limited effect on epigenetic marks of DNA obtained from blood., This work was funded by Ministerio de Ciencia e Innovación (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”); ACRONYM: NUPROVI
Alba Martín gratefully acknowledges receipt of a pre-doctoral grant (PRE2019-089288) from Ministerio de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033, “El FSE invierte en tu futuro”); CSIC is acknowledged for supporting Open Access publication., 3_****_L001_R1_001.fastq 3_****_L001_R2_001.fastq 4_****_L001_R1_001.fastq 4_****_L001_R2_001.fastq 16_****_L001_R1_001.fastq 16_****_L001_R2_001.fastq 17_****_L001_R1_001.fastq 17_****_L001_R2_001.fastq 20_****_L001_R1_001.fastq 20_****_L001_R2_001.fastq 21_****_L001_R1_001.fastq 21_****_L001_R2_001.fastq 29_****_L001_R1_001.fastq 29_****_L001_R2_001.fastq 30_****_L001_R1_001.fastq 30_****_L001_R2_001.fastq 31_****_L001_R1_001.fastq 31_****_L001_R2_001.fastq 32_****_L001_R1_001.fastq 32_****_L001_R2_001.fastq, No

7 páginas, 2 figuras, 2 tablas., Feed restriction during the early life of ewe lambs (F0) triggers the transfer of epigenetic marks to the next generation, thus impairing the reproductive performance of F1. However, the effects of this factor on milk production and composition, including its abundance in regulatory miRNA (many of which are contained in exosomes, small vesicles of endocytic origin that play a role in the modulation of immune response of the offspring) has not been tested so far. Therefore, in this study, the replacement ewe lambs (F0) obtained in a previous project (a group of ewes fed milk replacer ad libitum –ADL– vs. a group of ewes restricted –RES– to 62.5 % the intake level of milk replacer during the suckling period) were raised under similar post-weaning conditions and mated to obtain the progeny (F1). The F1 female lambs were also mated to obtain F2. Milk production was recorded during the peak lactation period of F1, and milk samples were obtained for each gland (udder half) separately to measure chemical composition, somatic cell counts (SCC), and bacteria counts. Moreover, exosomes were also isolated from the milk of each gland separately to obtain the miRNAome following a Next Generation Sequencing approach. No significant differences were found in either milk production of F1, chemical composition of milk (fat, protein, lactase), or bacteriology (colony forming units, CFU). However, SCC was reduced significantly in milk samples of F1-RES dairy sheep, whereas the abundance of five miRNAs was also modified. Thus, oar-miR-150, oar-miR-221, oar-miR-23a, oar-miR-27a, oar-miR-376c were all down modulated in F1-RES when compared to F1-ADL. Most of these miRNAs have been found to play a role in biological functions such as development, apoptosis, muscle differentiation, reproduction, or milk production. However, the exosomes extracted from the milk of these sheep (F1-RES) did not affect the production of IL-9 and IL-2 cytokines after in vitro culture with CaCo-2 cells. This study reveals that nutritional programming events such as feed restriction may drive the abundance of not only SCC but also some milk's bioactive components such as miRNAs, although it is not clear if these changes may modulate the immune response at the intestinal level of the offspring., This work was funded by Ministerio de Ciencia e Innovacion ´ (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033,
“FEDER, Una manera de hacer Europa”). Alba Martín gratefully acknowledges receipt of a pre-doctoral grant (PRE2019-089288) from
Ministerio de Ciencia e Innovacion ´ (MCIN/AEI/10.13039/501100011033, “El FSE invierte en tu futuro”). CENSYRA is also gratefully
acknowledged for milk analyses, Peer reviewed

5 páginas, 1 tabla.-Trabajo presentado al: XLVII Congreso Nacional y XXIII Internacional de la Sociedad Española de Ovinotecnia y Caprinotecnia (SEOC). pp. 74-77. Valencia (España). 18-20 de septiembre., Este trabajo fue financiado por el Ministerio de Ciencia e Innovación (PID2021-
126489OB-I00, MCIN/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”).
Alba Martín disfrutó de un contrato predoctoral (PRE2019-089288) financiado por el Ministerio
de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033, “El FSE invierte en tu futuro”)., Peer reviewed

Methionine supplementation during the suckling period of ewe lambs (F0) may modify DNA methylation in the germline (F0) during this critical window period for the neonate, hence promoting inter-generational transmission of epigenetic marks to the offspring (F1). This factor may alter the expression of specific genes and physiological traits throughout life of the offspring (F1). The results of the present study revealed that methionine supplementation during the suckling period of ewe lambs (F0) caused the transmission of epigenetic marks to the offspring (DNA obtained from the blood of 1-month old Assaf ewe lambs, F1), so genes involved in Developmental process and Anatomical structure development were differentially methylated in F1-MET when compared to F1-CTRL ewe lambs. Therefore, although the effects on feed efficiency during adulthood were limited, F1-MET dairy sheep showed a reduced live body weight during the whole life., This work was funded by Ministerio de Ciencia e Innovación (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”); ACRONYM: NUPROVI
Alba Martín gratefully acknowledges receipt of a pre-doctoral grant (PRE2019-089288) from Ministerio de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033, “El FSE invierte en tu futuro”); CSIC is acknowledged for supporting Open Access publication., Relationship between files, if important: S_1018_1.fastq (control group, F1-CTRL) S_1018_2.fastq (control group, F1-CTRL) S_1020_1.fastq (control group, F1-CTRL) S_1020_2.fastq (control group, F1-CTRL) S_1022_1.fastq (control group, F1-CTRL) S_1022_2.fastq (control group, F1-CTRL) S_1025_1.fastq (control group, F1-CTRL) S_1025_2.fastq (control group, F1-CTRL) S_1026_1.fastq (control group, F1-CTRL) S_1026_2.fastq (control group, F1-CTRL) S_1033_1.fastq (methionine group, F1-MET) S_1033_2.fastq (methionine group, F1-MET) S_1038_1.fastq (methionine group, F1-MET) S_1038_2.fastq (methionine group, F1-MET) S_1042_1.fastq (methionine group, F1-MET) S_1042_2.fastq (methionine group, F1-MET) S_1043_1.fastq (methionine group, F1-MET) S_1043_2.fastq (methionine group, F1-MET) S_1045_1.fastq (methionine group, F1-MET) S_1045_2.fastq (methionine group, F1-MET), Peer reviewed

16 páginas, 2 figuras, 6 tablas., Nutritional strategies that supply methyl donors, such as methionine in the early post-natal period of lambs could program feed efficiency, milk production and metabolism along the whole life due to persistent changes in epigenetic marks. To test this hypothesis, 34 newborn ewe lambs from the same flock were stratified and distributed into two groups with equal body weight distribution. The control group (F0-CTRL, n = 17) was fed ad libitum with a milk replacer. In contrast, the second group (F0-MET, n = 17) received the same milk replacer supplemented with 1 g methionine/kg DM. After weaning, all animals were raised the same way and fed ad libitum with a complete pelleted diet formulated according to nutrient requirements. At 45 days of age, blood was collected from the jugular vein for DNA isolation, and DNA methylation was analyzed by Reduced Representation Bisulfite Sequencing (RRBS) in ten animals [5 ewe lambs (F0-MET) vs. 5 ewe lambs (F0-CTRL), corresponding to 5 twin births, of which one lamb was assigned to each group]. The main results show that CPT1B, a gene involved in fatty acid catabolism, was differentially methylated in F0-MET lambs (45 days of life). In addition, these animals presented increased insulin and non-esterified fatty acid concentrations while cholesterol levels were reduced, most of these changes being persistent along the whole life. No differences were observed in milk production and feed efficiency during lactation. Still, the milk's fatty acid (FA) profile showed increased levels of long-chain fatty acids, n3 and n6 polyunsaturated fatty acids. All these results suggest that methionine supplementation in early life can modify ewes' milk fatty acid profile without compromising feed efficiency. However, it is necessary to clarify whether this effect is passed on to the offspring (F1) through epigenetic marks in the germline., This work was funded by Ministerio de Ciencia e Innovacion ´ (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033,
"FEDER, Una manera de hacer Europa"). Alba Martín gratefully acknowledges receipt of a pre-doctoral grant (PRE2019-089288) from
Ministerio de Ciencia e Innovacion ´ (MCIN/AEI/10.13039/501100011033, "El FSE invierte en tu futuro". The metabolomics analysis was
carried out at the UNI-TECH "OMICS" platforms of the Universita ` degli Studi di Milano. We acknowledge Dr. Manuela Fontana’s
necessary support from the UNI-TECH "OMICS" platforms of the Universita ` degli Studi di Milano. CENSYRA (Leon, ´ Spain) is also
gratefully acknowledged for milk analyses., Peer reviewed

Methionine supplementation during the suckling period of ewe lambs (F0) might program persistent changes on DNA methylation, thus modifying miRNA profile of colostrum exosomes during the lactation period when these animals are adults. Identifying these changes is important because may drive some effects on the offspring (F1) under natural rearing conditions. The results of the present study revealed changes in miRNA profile of colostrum exosomes as a consequence of methionine supplementation during the suckling period of ewe lambs (F0)., This work was funded by Ministerio de Ciencia e Innovación (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”); ACRONYM: NUPROVI
Alba Martín gratefully acknowledges receipt of a pre-doctoral grant (PRE2019-089288) from Ministerio de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033, “El FSE invierte en tu futuro”); CSIC is acknowledged for supporting Open Access publication., 1M_L001_R1_001.fastq.gz (F0-MET) 1M_L001_R2_001.fastq.gz (F0-MET) 8C_L001_R1_001.fastq.gz (F0-CTRL) 8C_L001_R2_001.fastq.gz (F0-CTRL) 19C_L001_R1_001.fastq.gz (F0-CTRL) 19C_L001_R2_001.fastq.gz (F0-CTRL) 28M_L001_R1_001.fastq.gz (F0-MET) 28M_L001_R2_001.fastq.gz (F0-MET) 31M_L001_R1_001.fastq.gz (F0-MET) 31M_L001_R2_001.fastq.gz (F0-MET) 35C_L001_R1_001.fastq.gz (F0-CTRL) 35C_L001_R2_001.fastq.gz (F0-CTRL), Peer reviewed

19 páginas, 8 figuras, 2 tablas., New therapeutic molecules for farm animals are needed to address worldwide problems in the food industry, like the rise of resistance among ruminant parasites and pathogenic microbes. Since in vivo testing would involve an excessive number of animals, with consequent ethical and economic issues, the generation of sheep intestinal organoids represents a promising close-to-reality in vitro model for veterinary drug development; however, the characterization and application of such organoids remain limited. In this study, ovine intestinal organoids were generated from adult LGR5+ stem cells from the intestinal crypts of freshly slaughtered lambs, and developed in an in vitro culture system. Morphological analysis via brightfield microscopy and immunocytochemical staining revealed a pseudostratified epithelium with multiple cell types, and distinct apical–basal polarity, while RNA sequencing validated the preservation of the physiological characteristics of the original organ. The development and characterization of a robust and reproducible protocol for culturing sheep duodenum intestinal organoids in a high-throughput screening (HTS) compatible format demonstrated reliability in HTS applications, with Z’-factor tests indicating robust assay performance. Dose–response studies using pre-identified compounds showed comparable pharmacodynamic profiles between mouse and sheep organoids. These findings establish sheep intestinal organoids as an innovative tool for veterinary pharmacology and toxicology, offering a cost-effective and sustainable platform to address challenges such as drug resistance and improve livestock health., This research was part of projects PID2020-119035RB-I00 and PID2021-126489OB-I00,
funded by Ministerio Ciencia, Innovación y Universidades (MICIU)/AEI/10.13039/501100011033,
“FEDER, Una manera de hacer Europa”. This research also received funds from University of León
and Fundación General de la Universidad de León y de la Empresa (FGULEM) through the ULE-PoC
(PRUEBAS DE CONCEPTO DE LA UNIVERSIDAD DE LEÓN, Plan TCUE 2023-2027) programme.
Grant PRE2021-096909 was funded by MICIU/AEI/10.13039/501100011033 and by “ESF Investing
in your future”., Peer reviewed

Adequate management of replacement ewe lambs (F0) in dairy sheep farms during postnatal life may modify the germline cells, thus promoting transmission of intergenerational effects to the offspring (F1). To test this hypothesis, 18 newborn male lambs (F1), either born from methionine-supplemented ewe lambs (F0 ewe lambs being fed ad libitum with a milk replacer supplemented with 1 g methionine/kg DM) or not supplemented (F0 ewe lambs being fed ad libitum with the same milk replacer with no methionine added), were included in the present study. All the male F1 lambs were managed exactly in the same way along the whole lifespan in order to bring out the differences caused by methionine supplementation of F0 dams. Our data show that the methionine supplementation of dams (F0) during the suckling period did not promote significant (p > 0.05) changes on feed intake, growth rate, or feed efficiency of F1 male lambs during the fattening period. Moreover, the meat chemical composition (proximal, fatty acid profile, and volatile compounds) was similar for both groups (p > 0.05), but the meat of F1-MET lambs presented higher redness and hardness (p < 0.05) when compared to F1-CTRL lambs. The biochemical profile also highlighted significant (p < 0.05) differences in the serum creatinine and calcium content that may be at least partially related to the meat quality traits observed. Overall, all these results suggest that methionine supplementation of lambs (F0) during early postnatal life causes permanent changes in the offspring. This has positive effects, such as achieving a more attractive color of lamb meat (F1) for consumers, and negative effects, such as reduced meat tenderness., This work was funded by Ministerio de Ciencia e Innovación (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”). Alba Martín gratefully acknowledges receipt of a pre-doctoral grant (PRE2019-089288) from Ministerio de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033, “El FSE invierte en tu futuro”)., Peer reviewed

This article belongs to the Special Issue Intestinal 3D Culture, Organoids and Gut-on-Chip in Intestinal Disorders, Ovine colostrum exosomes obtained from nutritionally programmed dairy ewes (F0) may present modifications in microRNAs, thus having consequences for the intestinal barrier function and immunity parameters of lambs (F1). To test this hypothesis, colostrum exosomes from two ewe groups [F0-MET (nutritionally programmed ewes being fed methionine during early life) and F0-CTRL (ewes not supplemented with methionine during early life)] were sequenced to compare differences in the miRNAome. In addition, these exosomes were added to an in vitro co-culture in a Transwell chamber system consisting of ovine duodenum intestinal organoids and macrophages to assess the expression of genes encoding tight junction proteins in organoids and immunity parameters in macrophages. Finally, the concentrations of cytokines (e.g., IL-12 and IL-6) were assessed by ELISA kits in the supernatants of the chamber containing macrophages. According to the miRNAome, the expression of two miRNAs (e.g., oar_miR_376c_3p and oar_miR_432) was reduced in the colostrum exosomes obtained from dairy ewes nutritionally programmed with dietary supplementation of methionine during early life (F0-MET ewes). These changes did not seem to modify the expression of intestinal barrier and immune response marker genes when these exosomes were added to a co-culture of ovine intestinal organoids and macrophages. However, the levels of IL-12 produced by macrophages were reduced (p < 0.05), which suggests the inhibition of inflammatory pathways. Further studies using ovine colostrum exosomes obtained from nutritionally programmed ewes will help to clarify their potential to improve the health of suckling lambs., This work was supported by grant PID2021-126489OB-I00 funded by MCIN/AEI/10.13039/501100011033, “FEDER Una manera de hacer Europa”, and by grant PID2020-119035RB-I00 funded by MICIN/AEI/10.13039/501100011033. G. Galli received grant PRE2021-096909 funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”., Peer reviewed

This article belongs to the Special Issue Organoid Models in Host–Pathogen Interactions and Disease Pathogenesis, [Institutional Review Board Statement] The animal study protocol was approved by the Ethics Committee of the University of León, protocol code OEBA-ULE-010-2023, in compliance with Spanish and EU legislation (RD 53/2013 and 2010/63/EU, approved on 14 April 2023)., The development of new alternative models is essential to overcome the limitations of traditional two-dimensional (2D) cell cultures and animal models. Three-dimensional (3D) models, such as organoids, better mimic the structural and functional complexity of mammalian organs, thereby reducing the ethical and economic issues related to animal experimentation. These systems provide more physiologically relevant environments, improving the accuracy of disease modeling and drug response prediction. In this context, we have developed mouse hepatic organoids from livers of adult BALB/c mice and characterized them by microscopy and transcriptional analysis. This model was applied to a robust and reproducible high-throughput screening (HTS) platform for testing cytotoxicity at the preclinical stage of drug discovery. In addition, mouse hepatic organoids were co-cultured with amastigotes of Leishmania donovani parasites to establish a model of host–parasite interaction, which was characterized by RNA-seq linked to differential expression analysis and cytokine production by the hepatic organoids. The findings provided in this work establish mouse hepatic organoids as an alternative model for drug discovery and pathogenesis studies., This research was funded by projects PID2020-119035RB-I00, PID2020-119031RB-I00, PID2021-126489OB-I00, funded by Ministerio de Ciencia, Innovación y Universidades (MICIU)/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”, and HaloSheep (Agroecological sheep/goat production system based on the valorization of halophytes of saline areas in the Mediterranean basin) funded under the PRIMA Programme by the European Union (NextGenerationEU) and Ministerio de Ciencia, Innovación y Universidades (PCI2022-132925). G.G. received Grant PRE2021-096909 funded by MICIU/AEI/10.13039/501100011033 and by “ESF Investing in your future”. M.-C.G.-M. received a PhD fellowship from Universidad de León., Peer reviewed

This article belongs to the Special Issue Feed Evaluation and Management for Ruminant Nutrition., The effects of pelleting a total mixed ration (TMR) for dairy sheep were investigated in an experiment involving 24 lactating Assaf ewes, which were assigned to two groups and fed the same TMR ad libitum, offered either in pelleted (PTMR group, n = 12) or in unpelleted form (CTMR group, n = 12). The experiment lasted 28 days, during which feed intake, eating behavior (including meal frequency and size, meal duration, eating rate, between-meal interval), and milk yield were recorded daily. Body weight (BW) was recorded on days 1 and 28 and milk samples were collected on days 1, 8, 15, 22 and 28 for milk composition analysis. Blood acid-base status was determined at the beginning and at the end of the trial. Ewes fed the CTMR diet exhibited (p < 0.05) a higher meal frequency and longer meal duration, along with a smaller meal size and slower eating rate. However, feed intake in this group was less than that in ewes fed PTMR only during the final two weeks of the experimental period. Total eating time was also longer (p < 0.001) in the CTMR group, whereas the average time between meals was shorter (p < 0.002). No differences (p > 0.05) were observed between dietary treatments in blood acid-base status, milk yield or milk composition. However, a diet x day interaction (p < 0.05) was detected for milk yield, as during the last 2 weeks of the experimental period the ewes fed the PTMR yielded more milk than those fed the CTMR. Feed conversion ratio did not differ between groups (p > 0.05), but body weight loss was greater in ewes fed the CTMR diet (−3.00 vs. −0.58 kg; p < 0.05). A trend toward improved feed efficiency was observed in the PTMR group when calculated based on milk yield corrected for that theoretically derived from the mobilization of body reserves (1.98 vs. 1.41 g DMI/kg milk; p = 0.077), with estimated contributions from body reserves of 485 g/day in the CTMR group and 70 g/day in the PTMR group. In conclusion, the use of pelleted total mixed rations in high-yielding dairy ewes enhances feed intake, feed efficiency, milk yield, and energy balance without adversely affecting milk composition or animal health in the short term., This work was funded by Ministerio de Ciencia e Innovación (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”)., Peer reviewed

This work was funded by Ministerio de Ciencia e Innovación (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”); ACRONYM: NUPROVI
Alba Martín gratefully acknowledges receipt of a pre-doctoral grant (PRE2019-089288) from Ministerio de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033, “El FSE invierte en tu futuro”); CSIC is acknowledged for supporting Open Access publication., File list:H1_1001_R1.fastq; H1_1001_R2.fastq; H2_1001_R1.fastq; H2_1001_R2.fastq; H3_1001_R1.fastq; H3_1001_R2.fastq; H4_1001_R1.fastq; H4_1001_R2.fastq; H5_1001_R1.fastq; H5_1001_R2.fastq; ORG1_1001_R1.fastq; ORG1_1001_R2.fastq; ORG2_1001_R1.fastq; ORG2_1001_R2.fastq; ORG3_1001_R1.fastq; ORG3_1001_R2.fastq; ORG4_1001_R1.fastq; ORG4_1001_R2.fastq; ORG5_1001_R1.fastq; ORG5_1001_R2.fastq;, Peer reviewed

Twenty-eight pregnant Merino ewes were used to study the effect of including 4 g/kg of betaine in a complete pelleted ration on feed intake and animal performance of both, dams (F0) and female offspring (F1), including the first lactation period. To this end, fourteen ewes (F0-CTL group) received a complete pelleted ration (control diet) ad libitum during the last two months of gestation. The remaining animals (F0-BET group; n = 14) received the same diet, but with the addition of 4 g/kg of betaine. The results of the present study revealed that betaine supplementation during the gestation period of Merino ewes caused differentially methylated regions (DMRs) in the female offspring that were assessed at 53,649 and 2164 sites using MethylKit (q value < 0.05) and CGmap Tools (FDR < 0.05), respectively., This work was funded by Ministerio de Ciencia e Innovación (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”); ACRONYM: NUPROVI
CSIC is acknowledged for supporting Open Access publication., File list: S_1006_1.fastq; S_1006_2.fastq; S_1010_1.fastq; S_1010_2.fastq; S_1015_1.fastq; S_1015_2.fastq; S_1020_1.fastq; S_1020_2.fastq; S_1023_1.fastq; S_1023_2.fastq; S_1055_1.fastq; S_1055_2.fastq; S_1064_1.fastq; S_1064_2.fastq; S_1065_1.fastq; S_1065_2.fastq; S_1066_1.fastq; S_1066_2.fastq; S_1074_1.fastq; S_1074_2.fastq;, Peer reviewed

13 páginas, 2 figuras, 6 tablas., Methionine supplementation during the suckling period of ewe lambs may modify DNA
methylation in the germline (F0) during this critical window period for the neonate. This
circumstance may promote the intergenerational transmission of epigenetic marks to the
offspring (F1), thus altering the expression of specific genes and physiological traits throughout
F1 life. To test this hypothesis, 27 newborn ewe lambs (F1) born from either ewes being supplemented 0.1 % methionine (dry matter basis) during the suckling period (F0-MET) or not
supplemented (F0-CTRL) were assigned to two different groups (F1-CTRL, n = 13 and F1-MET,
n = 14), being the dietary treatment of their mothers (F0) the only source of variation. Thus,
all the F1 animals (both groups) were raised exactly in the same way along the whole life
(including lactation). In this study, we determined differences in the global blood methylation
patterns, biochemical profile, and metabolome of female offspring (F1). Our data showed that
functional categories such as those related to developmental process and anatomical structure
development were significantly enriched in the F1-MET ewe lambs due to differentially methylated regions of genes in these categories. These F1-MET ewes also presented lower live body
weight (P < 0.05) and reduced lipomobilization and milk yield (P = 0.099) during the lactation
period, together with increased PUFAs content (P = 0.075) in the milk fatty acid profile when
compared to F1-CTRL. Increased levels of insulin (P = 0.031) and β-hydroxybutyrate (P = 0.043),
along with certain features of the metabolome profiles, revealed altered lipid metabolism when compared to F1-CTRL animals. As far as fecal microbiota is concerned, no significant differences
were found in alpha and beta diversity between the two groups. However, DESeq2 analysis
performed on OTU-filtered data revealed that Treponema (genus) and Spirochaetaceae (family),
both from the Spirochaetes phylum, were reduced in the F1-MET group compared to the F1-CTRL
group (log2FC=-3.96, padj < 0.05). The results suggest that early methionine supplementation in
F0 ewe lambs has an intergenerational impact on their F1 female offspring, with negative consequences on lipomobilization and milk production during F1 lactation., This work was funded by Ministerio de Ciencia e Innovacion ´ (PID2021–126489OB-I00, MCIN/AEI/10.13039/501100011033,
“FEDER, Una manera de hacer Europa”). CENSYRA is also gratefully acknowledged for milk analyses., Peer reviewed

16 páginas, 6 figuras, 3 tablas., The liver coordinates metabolic processes that determine growth, health, and production efficiency in livestock species. In ruminants, hepatic metabolism is adapted to support glucose homeostasis, lipid partitioning, and nitrogen utilization during ruminal fermentation. Therefore, predictive experimental models are essential for studying nutrient-mediated regulation of liver function. However, conventional two-dimensional hepatocyte cultures fail to reproduce the structural organization and functional complexity of native liver tissue, limiting their translational value., The author(s) declared that financial support was received for this
work and/or its publication. This work was funded by Ministerio de
Ciencia e Innovación (PID2021-126489OB-I00, MCIN/
AEI/10.13039/501100011033, “FEDER, Una manera de hacer
Europa”)., Peer reviewed

The effects of pelleting a total mixed ration (TMR) for dairy sheep were investigated in an experiment involving 24 lactating Assaf ewes, which were assigned to two groups and fed the same TMR ad libitum, offered either in pelleted (PTMR group, n = 12) or in unpelleted form (CTMR group, n = 12). The experiment lasted 28 days, during which feed intake, eating behavior (including meal frequency and size, meal duration, eating rate, between-meal interval), and milk yield were recorded daily. Body weight (BW) was recorded on days 1 and 28 and milk samples were collected on days 1, 8, 15, 22 and 28 for milk composition analysis. Blood acid-base status was determined at the beginning and at the end of the trial. Ewes fed the CTMR diet exhibited (p < 0.05) a higher meal frequency and longer meal duration, along with a smaller meal size and slower eating rate. However, feed intake in this group was less than that in ewes fed PTMR only during the final two weeks of the experimental period. Total eating time was also longer (p < 0.001) in the CTMR group, whereas the average time between meals was shorter (p < 0.002). No differences (p > 0.05) were observed between dietary treatments in blood acid-base status, milk yield or milk composition. However, a diet x day interaction (p < 0.05) was detected for milk yield, as during the last 2 weeks of the experimental period the ewes fed the PTMR yielded more milk than those fed the CTMR. Feed conversion ratio did not differ between groups (p > 0.05), but body weight loss was greater in ewes fed the CTMR diet (−3.00 vs. −0.58 kg; p < 0.05). A trend toward improved feed efficiency was observed in the PTMR group when calculated based on milk yield corrected for that theoretically derived from the mobilization of body reserves (1.98 vs. 1.41 g DMI/kg milk; p = 0.077), with estimated contributions from body reserves of 485 g/day in the CTMR group and 70 g/day in the PTMR group. In conclusion, the use of pelleted total mixed rations in high-yielding dairy ewes enhances feed intake, feed efficiency, milk yield, and energy balance without adversely affecting milk composition or animal health in the short term., This work was funded by Ministerio de Ciencia e Innovación (PID2021-126489OB-I00, MCIN/AEI/10.13039/501100011033, “FEDER, Una manera de hacer Europa”)., info:eu-repo/semantics/publishedVersion