Male yaks adapt to heat stress with enhancement of immunomodulation, anti-oxidation, and blood oxygen delivery.

Yaks adapt to extremely low temperatures, but they are more susceptible to heat stress (HS). The adaptive mechanisms with crucial plasma protein markers regulating the response to HS remain elusive. In this study, data-independent acquisition proteomics were used to evaluate the thermal adaptability under chronic HS and thermal-neutral conditions. As a result, yaks increased body temperatures and respiratory rates in response to HS. Eight differential proteins mainly related to vasodilatation were decreased by HS, but another four proteins associated with blood oxygen delivery were presented at higher levels. Complement and coagulation cascades pathway was activated by HS, and more proteins were upregulated to protect against inflammation and oxidative stress by higher levels of antioxidant proteins. It is likely that yaks react to HS with enhancement of immunomodulation, anti-oxidation, and blood oxygen delivery, which is conducive to taking appropriate environment and nutrition management strategies to get healthy and high-performing yaks in low-altitude regions during summer.

Physiological and Metabolic Adaptation to Heat Stress at Different Altitudes in Yaks.

Yaks have strong adaptability to extremely cold and hypoxic conditions but are susceptible to high ambient temperature when yaks are raised in low-altitude areas during the high-temperature season. Twenty-four adult male yaks with similar weights and ages were randomly divided into TN (Thermoneutral, altitude = 3464 m), LHS (Light heat stress, altitude = 1960 m), and MHS (Medium heat stress, altitude = 906 m) groups to evaluate adaptation strategies to HS. Non-targeted and targeted metabolomics were applied to investigate the effects of different extents of HS on yaks. LHS- and MHS-yaks showed higher rectal temperatures and respiratory rates than TN-yaks. MHS-yaks had higher levels of red blood cells (RBCs), hemoglobin (Hb), whole blood relative index of middle shear at a shear rate of 5 S-1 (WMS), whole blood relative index of high shear at a shear rate of 200 S-1 (WHS), Casson viscosity (CV), middle shear flow resistance at a shear rate of 5 S-1 (MSFR), and high shear flow resistance at a shear rate of 200 S-1 (HSFR) as compared to TN- and LHS-yaks. Differential metabolites and metabolic pathways, including fatty acid metabolism, lipid metabolism, glucose metabolism, and amino acid metabolism, were altered by HS. Metabolites in the glucose metabolism pathway in LHS- and MHS-yaks were lower than those in TN-yaks. However, LHS-yaks showed higher levels of metabolites in the HIF-1 signaling pathway compared to TN- and MHS-yaks. Most of the tricarboxylic acid cycle (TCA) intermediates and fatty acids were significantly decreased in MHS-yaks compared to the other two groups. As a whole, yaks raised at a low altitude (25.6 °C) suffered from severe HS, but they adapted to HS with vasodilatation for dissipating heat and the increased antioxidants and metabolite levels of energy substrates.

Physiological and Proteomic Responses of Dairy Buffalo to Heat Stress Induced by Different Altitudes.

Buffalo are mainly distributed in low-altitude (LA), medium-altitude (MA), and high-altitude (HA) regions characterised by different thermal and oxygen environments in Yunnan province, China. Due to black skin, sparse hair, and the low density of skin sweat glands, buffalo are more sensitive to heat stress. Here, we used data-independent acquisition (DIA) proteomics to reveal a broad spectrum of proteins that play roles in adaptation to the heat stress of buffalo raised at low altitude or hypoxia at high altitude. LA buffalo showed higher body temperatures than MA- and HA buffalo, and HA buffalo had higher levels of GSH and SOD and lower levels of ROS compared to LA and MA buffalo. In 33 samples, 8476 peptides corresponding to 666 high-confidence proteins were detected. The levels of circulating complement proteins in the immune pathways were lower in LA and MA buffalo than in HA buffalo. There were higher levels of alpha-1 acid glycoprotein in LA buffalo than in MA and HA buffalo. Relative to MA buffalo, levels of blood oxygen delivery proteins were higher in LA and HA buffalo. A higher abundance of apolipoproteins was detected in LA and MA buffalo than in HA buffalo. In summary, buffalo adopted similar adaptation strategies to oxidative stress induced by heat stress or hypoxia, including immunological enhancement, high efficiency of blood oxygen delivery, and the inhibition of lipid oxidation.

The use of metabolomics to reveal differences in functional substances of milk whey of dairy buffaloes raised at different altitudes.

Buffalo milk is nutrient-rich and contains less cholesterol than cow milk. Dairy buffaloes are widely distributed at different altitudes in the Yunnan Province, China; however, the impacts of altitude on the whey-derived functional metabolites of buffalo milk whey are not well understood. Here, we used non-targeted and targeted metabolomics to evaluate the differential metabolites in the milk whey of buffaloes raised at low altitudes (LA), medium altitudes (MA), and high altitudes (HA). ANOVA statistical test was performed to acquire differential metabolites using IBM SPSS statistics 22 software. The results showed that LA- and MA-milk whey had higher levels of amino acids (glutamine and pyroglutamic acid) and vitamin B6 than HA-milk whey. LA-milk whey had higher levels of the carbohydrates involved in galactose, amino sugar, and nucleotide sugar metabolism than MA- and HA-milk whey, but HA-milk whey showed significantly higher levels of free fatty acids. In conclusion, owing to the biological functions of their most abundant components, LA-milk is more suitable for the production of functional milk with high levels of amino acids, vitamin B6, and carbohydrates; while HA-milk is suitable as raw milk for the production of dairy products with high free fatty acid content.

DIA proteomics reveals hypotensive and immune-enhancing constituents in buffalo whey from different altitudes.

The selection of raw milk with high levels of functional components that have health-promoting activities is very important for the exploitation and production of functional milk, but the differences in the functional components of whey from buffalo raised at different altitudes have not been thoroughly investigated. Here, we detected the effects of altitudes on the functional components in whey from dairy buffalo farms situated at low altitude (LA), medium altitude (MA), and high altitude (HA) sites with data-independent acquisition proteomic approaches. In 33 samples, 9331 peptides corresponding to 1008 high-confidence proteins were detected. HA-whey had a lower level of angiotensinogen than that of the LA- and MA-whey, and conversely contained higher levels of immune-enhancing components than for the latter two groups. Differential proteins were involved in vascular smooth muscle contraction, complement and coagulation cascades, and the secretion, production and regulation pathways in immune components. LA-whey showed higher levels of lymphocyte antigen and selenoprotein F than that of the HA-whey. Owing to the biological functions of their most abundant components, HA- and LA-whey are suitable for the processing of functional milk for lowering blood pressure, and the production of immune milk, respectively.

Serum proteomics analysis reveals the thermal fitness of crossbred dairy buffalo to chronic heat stress.

Chronic heat stress (CHS) reduces the production efficiency of the buffalo dairy industry. Relatively low-abundance proteins with particular functions in biological processes are changed by CHS. The present study aimed to quantify the differences in low-abundance proteins of crossbred dairy buffaloes under CHS and thermal-neutral (TN) conditions. With label-free quantification, 344 low-abundance proteins were identified in serum. Of these, 17 differentially expressed low-abundance proteins with known functions were detected, and six of the differentially expressed proteins related to heat stress were validated with parallel reaction monitoring. Lipase (LPL), glutathione peroxidase 3 (GPX3), cathelicidin-2 (CATHL2), ceruloplasmin (CP), and hemoglobin subunit alpha 1 (HBA1) cooperatively played roles in the thermal fitness of dairy buffalo by decreasing heat production and increasing blood oxygen delivery. Also, dairy buffaloes may adapt to CHS and hypoxia with high levels of RBCs, HBA1 and CP to increase blood oxygen delivery capacity.

Impacts of drainage beds covered with sand and wood shavings on the comfort behaviour of dairy buffalo in a temperate climate.

BACKGROUND: Comfortable beds play an important role in increasing the ruminant and milk production efficiency of dairy buffalo. In loose housing systems, cow lying comfort depends on both the bedding materials and bed base. RESULTS: Buffaloes spent more lying time on sand beds at depth of 15 and 20 cm (S-15 and S-20) than on beds of 10 cm (S-10a) beds or in the feed alley in Exp1 (P < 0.01). No significant difference in the cow comfort index (CCI) was detected between S-10a and S-15; however, both showed higher CCI than that of the S-20 bed. In Exp2, buffaloes spent more time lying on the wood shavings at depth of 15 cm (WS-15) bed followed by the WS-20 and S-10b beds, respectively (P < 0.01), and CCI was greater in the WS-15 bed than in the S-10b and WS-20 beds (P < 0.05). CONCLUSION: A depth of sand or wood shavings at 15 cm can meet the lying comfort requirements of dairy buffaloes when bedding materials are used above drainage beds.

Effects of surface materials of self-draining beds on cattle behavior in a temperate climate.

OBJECTIVE: The objective of the present experiment was to construct self-draining beds to keep surface bedding materials clean and dry for beef cattle comfort in a temperate climate. METHODS: In Experiment 1, a self-draining bed was covered with sand at depths of 10 cm (S-10a), 15 cm (S-15), and 20 cm (S-20) respectively. In Experiment 2, self-draining beds of different sizes were covered with 10 cm of sand (S-10b) and wood shavings (WS) at depths of 15 cm and 20 cm (WS-15 and WS-20). Fifteen cattle were engaged to evaluate the comfort of self-draining beds covered with different bedding materials. RESULTS: No cattle lay in the feed alley and cattle spent more time lying on S-10a than S-15 or S-20 in Experiment 1 (p<0.01). No difference in lying time was detected between S-15 and S-20 (p>0.05). In Experiment 2, no cattle selected the feed alley as the lying area. Cattle preferred WS-15 as the lying area and time spent lying on WS-20 was slightly higher than on S-10b (p<0.05). Feces weight was higher in the feed alley than in the different bedding areas in both Experiments 1 and 2 (p<0.01). CONCLUSION: Sand-bedding depth at 10 cm and WSs at 15 cm above the self-draining bed can provide for the lying comfort of beef cattle. Design of a special feed alley to hold most of the feces to keep bedding materials clean and dry is desirable for organic beef cattle in a loose barn.

Directed modification of a ruminal cellulase gene (CMC-1) from a metagenomic library isolated from Yunnan gayal (Bos frontalis).

Gayal (Bos frontalis) of the Yunnan region is well adapted to harsh environmental conditions. Its diet consists predominantly of bamboo, reeds, and woody plants, suggesting that the rumen of this species contains many fiber-degrading bacteria and cellulases. The aim of this study was to identify and modify specific cellulases found in the gayal rumen. In the present study, a directed evolution strategy of error-prone PCR was employed to improve the activity or optimal temperature of a cellulase gene (CMC-1) isolated from gayal rumen. The CMC-1 gene was heterologously expressed in Escherichia coli (E. coli) BL21, and the recombinant CMC-1 protein hydrolyzed carboxyl methyl cellulose (CMC) with an optimal activity at pH 5.0 and 50 °C. A library of mutated ruminal CMC-1 genes was constructed and a mutant EP-15 gene was identified. Sequencing analysis revealed that EP-15 and CMC-1 belonged to the glycosyl hydrolase family 5 (GHF5) and had the highest homology to a cellulase (Accession No. WP_083429257.1) from Prevotellaceae bacterium, HUN156. There were similar predicted GH5 domains in EP-15 and CMC-1. The EP-15 gene was heterologously expressed and exhibited cellulase activity in E. coli BL21 at pH 5.0, but the optimum temperature for its activity was reduced from that of CMC-1 (50 °C) to 45 °C, which was closer to the physiological temperature of the rumen (40 °C). The cellulase activity of EP-15 was about two times higher than CMC-1 at 45 °C or PH 5.0, and also was more stable in response to temperature and pH changes compared to CMC-1. This study successfully isolated and modified a ruminal cellulase gene from metagenomics library of Yunnan gayal. Our findings may obtain a useful cellulase in future applications and present the first evidence of modified cellulases in the gayal rumen.

Metabolomics Reveals that Crossbred Dairy Buffaloes Are More Thermotolerant than Holstein Cows under Chronic Heat Stress.

Heat stress (HS) threatens the worldwide dairy industry by decreasing animal production performance and health. Holstein cows and dairy buffaloes are the most important dairy animals, but their differences in the metabolic mechanism of thermotolerance remain elusive. In this study, we used serum metabolomics to evaluate the differences in thermotolerance between Holstein cows and crossbred dairy buffaloes under chronic heat stress (HS) and thermal-neutral conditions. In response to HS, the body temperatures and respiratory rates were increased more for Holstein cows than for dairy buffaloes (38.78 vs 38.24 °C, p < 0.001; 43.6 vs 32.5 breaths/min, p < 0.001). HS greatly affected serum metabolites associated with amino acids, fatty acids, and bile acids. The enriched metabolic pathways of these serum metabolites are closely related to HS. We demonstrated that buffaloes adapt to HS by adopting a metabolism of branched-chain amino acids and ketogenic amino acids and gluconeogenesis, but Holstein cows decrease the effect of HS with citrulline and proline metabolism. Both physiological parameters and serum metabolic profiles indicate that dairy buffaloes are more thermotolerant than Holstein cows, providing the feasibility to vigorously develop the buffalo dairy industry in tropical and subtropical regions.

Isolation and cDNA characteristics of MHC-DRA genes from gayal (Bos frontalis) and gaytle (Bos frontalis × Bos taurus).

The mammalian major histocompatibility complex (MHC) plays important roles in pathogen recognition and disease resistance. In the present study, the coding sequence and the 5'- and 3'-untranslated regions of MHC class II DR alpha chain (the DRA gene) from rare gayal and gaytle were cloned and analyzed to dissect structural and functional variations. The nucleotide and amino acid sequences for the DRA genes in gayal (Bofr-DRA) and gaytle (Bofr × BoLA-DRA) were almost identical to those for cattle and yak (99%). Compared to yak, two amino acids substitutions in the signal peptide (SP) domain for gayal were found within all Bos animals. Except for only one replacement in the amino acid within the α2 domain of the DRA protein in gayal, the additional residues were highly conserved across the species investigated. The 20 peptide-binding sites (PBS) of Bofr-DRA and Bofr × BoLA-DRA were essentially reserved in the α1 domain among all species investigated. The lesser degree of substitution in Bofr-DRA is concordant with the concept that the DRA gene is highly conserved among all mammals. The very high degree of conservativity of the DRA gene among ruminants, including gayal, suggests its recent evolutionary separation.

Impacts of a freedom farrowing pen design on sow behaviours and performance.

The limited space in farrowing crate imposes many challenges, such as prolonged farrowing duration and high piglet stillbirth rate. Although the features of farrowing pens compensate for the drawbacks of farrowing crates, they are associated with high piglet crushing mortality caused by the greater space afforded to sows and their rolling-over behaviour. Therefore, a freedom farrowing pen was designed to overcome the drawbacks of both farrowing crates and farrowing pens. The main features of the freedom farrowing pen are its left anti-crushing bar and detachable right anti-crushing bar on the sides of the sow lying area. It also has a 10 cm-high anti-crushing bar in the non-lying area. Eighteen healthy, multiparous Yorkshire sows (3-7 parity) were averaged and randomly assigned to farrowing crates, farrowing pens, and freedom farrowing pens to compare the effects of the farrowing systems on sow behaviour and performance. Results showed that the farrowing duration and the mean piglet birth intervals were longer for the sows in farrowing crates than for those in farrowing pens and freedom farrowing pens (P<0.05), but there was no difference between the sows in farrowing pens and those in freedom farrowing pens (P>0.05). The piglet stillbirth rate was higher for the sows in farrowing crates than for those in farrowing pens and freedom farrowing pens (P<0.001). Crushing mortality was higher among piglets in farrowing pens (P<0.001), but there was no difference between piglets in freedom farrowing pens and those in farrowing crates (P>0.05). The freedom farrowing pen and the farrowing pen allowed sows to turn around and move freely, but because of the different structures of their anti-crushing bars, the increase in sow movement did not cause higher piglet crushing mortality (P>0.05). Sows in freedom farrowing pens were found to be more protective of their piglets.

Effects of neoprene mat on diarrhea, mortality and foreleg abrasion of pre-weaning piglets.

Modern commercial swine farrowing crates are typically equipped with slatted iron floor to improve management efficiency (e.g., ease of manure handling, cleanliness of the farrowing crates and hence improved animal hygiene). However, the bare and hard floor surface can impair the welfare of the sow-litter because of some undesirable impacts on the pigs, such as foreleg abrasion, large temperature gradients between the cold floor surface and the abdomen of the piglets (hence higher susceptibility to diarrhea), and higher pre-weaning mortality or morbidity. Although straw bedding has been shown to be conducive to providing better environment for the sow-litter, use of straw creates challenges in terms of economics, hygiene and manure handling. This study investigates the use of neoprene mat (NM) in key areas of the farrowing crates - underneath the sow and in the piglet suckling area to improve the microenvironment and hence welfare of the sow-litter. Two experiments were conducted, each involving 12 sow-litters. The first experiment was to evaluate the thickness of a rectangular-shaped NM (7, 10 or 13 mm) vs. the slatted iron floor (control or Ctrl) and collect the corresponding animal response data; while the second follow-up experiment was to verify the benefits of supplying an improved, double concave (or H)-shaped NM with 10mm thick (CNM10) vs. Ctrl for the farrowing operation. Results of both experiments demonstrated considerable benefits of the NM placement in the farrowing crates. Specifically, the NM reduced the piglet foreleg lesion area and joint swellings (0% for NM vs. 8-10% for Ctrl during suckling periods in both Expts 1 and 2, P<0.001); reduced pre-weaning piglet crushing mortality (18.5+/-5.0%, 6.7+/-3.3% and 9.1+/-5.2% and for Ctrl, NM7 and NM10 and in Expt 1, P<0.05); and reduced piglet diarrhea morbidity (0.6+/-0.2% for CNM10 vs. 2.7+/-0.3% for Ctrl in Expt 2, P<0.01). Piglets in the NM litters had smaller temperature gradients between the abdomen and the contact floor surface (3.8+/-2.3 degrees C for NM vs. 7.6+/-0.5 degrees C for Ctrl in Expt 1, P<0.001; 9.2+/-0.5 degrees C for CNM10 vs. 15.9+/-0.5 degrees C for Ctrl in Expt 2, P<0.001). Moreover, sows in the NM regimens showed longer transition time when changing from standing to lying position (7.4+/-0.3s for CNM10 vs. 4.5+/-0.2s for Ctrl in Expt 2, P<0.05), indicative of more floor comfort for the NM condition. Results of this study suggest that supply of NM underneath the sow and in the piglet suckling area is conducive to enhancing comfort, health and welfare of the sow and litter.