Introduction

Yak is an ancient even-toed ruminant animal residing in the Qinghai-xizang plateau and its surrounding regions with altitude over 3000 m1,2. As a results long time natural domestication, those herbivores own ability to adapted the harsh environments like extremely cold, anoxic, strong ultraviolet rays and shortage of pasture3. There are around 14–17 million yaks globally including countries of China, Nepal, Bhutan, India and others, and 90%of them are raised in China4. Yaks can provide nutritional milk and meat, high-quality fur, fermented manure, and low-cost fuel, and serve as a means of transport, which makes them critically important to local people in the cold plateau regions5. Chawula yak is mainly distributed in the Nyainrong County, Naqu, China, with a northern latitude of 32° 06′ and an east longitude of 92° 18′. Nyainrong County has an average altitude of more than 4700 m, with an annual average precipitation of 400 mm and a temperature of around 0 °C. Hence, highly efficient breeding of yaks is of great importance to local economic and farmer livings.

It is common sense that the nutritional supply of calves during the preweaning period is highly associated with the development of the gastrointestinal tract and immunity of animals6. The starter feed supplementation in yak calves significantly improves growth performance, promotes rumen and organ development, and alters the gut microbiota, thus starter feeds are generally more effective than traditional maternal grazing for productivity in barn-fed conditions. Therefore, products like milk replacer, starter, and alfalfa hay were produced to promote the growth of calves7. Calf starter feeds are composed of delicious and digestible ingredients, which are commonly employed for the transition of young ruminants from breast feeding to solid feeds8. The complete microbial community, namely microbiota, consists of trillions of microbes, including viruses, prokaryotes, and eukaryotes9, which is connected with the hosts’ digestive absorption, metabolism, intestinal barrier, and immunity10,11. The gut microbiota is dynamic (amount, vitality and composition) affected by factors of age, genetic variation, diet, lifestyle, medications and others12, which are related to the development and treatment of many diseases13. In the reorganization of the important functions of microbiota, more and more attentions were paid to the regulation of microbiota for finding novel therapies like faecal microbiota transplantation, specific additives, and probiotics14,15,16. Previous studies found that starter feeds promoted lambs, yaks and piglets’ growth via alliterating microbiota7,17,18. However, there is limited research about the effect of starter feeds on Chawula yak calves. Therefore, we carried out this experiment to disclose the impact of starter feeds on the growth, antioxidant ability and intestinal microbiota of early weaning Chawula yak calves to explore novel highly efficient breeding methods in plateau regions.

Results

Weight, body sizes and antioxidant ability of yak calves

The body weight of yak calves in DFT was signally higher than that of control animals in DFC (P < 0.05), and the net weight growth rate in DFO (P < 0.001) and DFT (P < 0.0001) were both obviously higher than that in the DFC group (Fig. 1a). DFT calves gained 4 kg more than control (DFC) group. The chest girth (bust) in group DFO (P < 0.05) and DFT (P < 0.05) were both markedly higher than that in the DFC group, where there was no observable difference of height, body length, body height, and circumference of cannon bone among calves in different groups (Fig. 1b). The serum contents of T-AOC in DFC calves were markedly lower than DFO (P < 0.01) and DFT (P < 0.001) yaks. Also, T-AOC was obviously higher in DFT yaks than in DFO animals (P < 0.05). The levels of GSH-Px were significantly higher in DFO (P < 0.01) and DFT (P < 0.01) yaks than DFC yaks. In contrast, no obvious difference was detected in SOD and MDA levels among yak calves in different groups (Fig. 1c).

Fig. 1
Fig. 1
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Weight, body sizes and antioxidant ability compared analysis of yak calves in different groups. (a) Body weight, (b) body size, (c) Antioxidant ability. Data are presented as SD, * P < 0.05, ** P < 0.01, *** P < 0.001, and *** P < 0.0001.

Sequencing data of yak calves in different groups

There were more than 47,000 (DFC > 55,700, DFO > 48,800, DFT > 47,500) raw and 45,000 (DFC > 51,500, DFO > 44,600, DFT > 45,000) filtered reads in yak calf (Table 1). There were 7396 ASVs in yak calves, with 193 shared ASVs in the three groups (Fig. 2a). There was no significant difference of the α-diversity index among different yak groups (Table 2, Fig. 2b). The rarefaction curves of all yak calves, indicating that those samples were sufficient to represent the bacterial diversity (Fig. 2c). All of the yaks’ rank abundance curves were gently in a horizontal direction, reflecting higher evenness in yaks (Fig. 2d).

Table 1 Statistical analysis of sequencing data of calves.
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Fig. 2
Fig. 2
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Venn map and alpha diversity analysis of calves in different groups. (a) Venn map, (b) Alpha diversity indexes, (c) Rarefaction curve, (d) Rank abundance curve.

Table 2 Statistical analysis of alpha diversity index in yak calves in different groups.
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Microbiota comparison analysis of yak calves in different taxonomic levels

In the phylum level, Bacteroidota and Firmicutes A were the primary phyla in DFC (66.10%, 23.84%), DFO (68.25%, 29.07%), and DFT (66.81%, 29.53%) (Fig. 3a). At the class level, Bacteroidia and Clostridia 258483 were the dominating classes in DFC (66.09%, 23.83%), DFO (68.25%, 29.06%) and DFT (66.82%, 29.52%) (Fig. 3b). At the order level, Bacteroidales and Oscillospirales were the main orders in DFC (66.35%, 9.52%) and DFO (69.84%, 13.43%), while Bacteroidales (52.39%) and Chitinophagales (15.90%) were the staple orders in DFT (Fig. 3c). At the family level, Bacteroidaceae and Muribaculaceae were the primary families in DFC (48.68%, 13.53%) and DFO (50.30%, 9.45%), while Bacteroidaceae (36.95%) and Saprospiraceae (17.23%) were the prominent families in DFT (Fig. 3d). At the genus level, the prime in different yak groups were Paraprevotella (47.27%) and CAG-485 (13.00) in DFC, Paraprevotella (42.25%) and Phocaeicola A 858004 (9.97%) in DFO, and Paraprevotella (26.41%) and OLB9 (20.63%) in DFT, respectively (Fig. 3e).

Fig. 3
Fig. 3
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Microbiota comparing analysis of yak calves in different taxa. (a) Phylum, (b) Class, (c) Order, (d) Family, (e) Genus.

Exploring biomarkers in yak calves in different groups

Β-diversity analysis showed that there a clear distance among the three yak calf groups via PCoA, NMDS, and UPGMA analysis (Fig. 4a–c), and PERMANOVA also confirmed it with an obvious difference (P = 0.002 < 0.01, Fig. 4d).

Fig. 4
Fig. 4
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β-diversity analysis of yak calves in different groups. (a) PCoA, (b) NMDS, (c) UPGMA, (d) PERMANOVA.

Heatmap showed that phyla of Firmicutes D, Firmicutes B 370539, Patescibacteria, Campylobacterota, Planctomycetota, Actinobacteriota, Gemmatimonadota, Chloroflexota, Desulfobacterota I, Nitrospirota A 437815, Desulfobacterota G 459546, Fusobacteriota, Bdellovibrionota E, Dormibacterota, Desulfobacterota E, Thermoproteota, Methanobacteriota A 1229, Eisenbacteria, Fibrobacterota, SAR324, Myxococcota A 473307, Proteobacteria, Acidobacteriota and Eremiobacterota were higher in DFC yak, and Spirochaetota, Bacteroidota, Firmicutes G and CSP1-3 were higher in DFO ruminants, while Synergistota, Chlamydiota, Elusimicrobiota, Halobacteriota, Marinisomatota and Cyanobacteria were higher in DFT animals (Fig. 5a). At the genus level, the abundance of Bacteroides H, Limosilactobacillus, Peptococcus, UBA6857, Acinetobacter, Brevundimonas, Faecalimonas, RUG420, Pygmaiobacter, Lactobacillus, CAG-488, CAG-485, CAG-269, Enterenecus, and Anaerobutyricum were higher in DFC groups. Jeotgalibaca, Corynebacterium, Paramuribaculum, RUG13077, Limivicinus, Bact-08, Alistipes A 871400, UBA4334, UBA737, Avispirillum, Faecousia and UBA5905 were higher in DFO group, while CCUG-7971, UMGS1994, CAG-273, OLB9, Turicibacter, Onthenecus, Limiplasma, Fimenecus, Copromorpha, Romboutsia B and SFMI01 were higher in the DFT group (Fig. 5b).

Fig. 5
Fig. 5
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Heatmap analysis of the top 50 species among yak calves in different groups. (a) Phylum, (b) Genus.

LEfSe showed that CAG 485 (P < 0.05), Acinetobacter (P < 0.05), Alloprevotella (P < 0.05) and CAG 485 (P < 0.05) were signally higher in DFC groups. Alistipes A 871400 (P < 0.01), Alloprevotella (P < 0.01), UBA4334 (P < 0.05), Faecousia (P < 0.05) and Acutalibacteraceae (P < 0.05) were obviously higher in DFO yaks. In contrast, SFMI01 (P < 0.05), CAG 273 (P < 0.05), OLB9 (P < 0.05), Akkermansia (P < 0.05), Cryptobacteroides sp902785575 (P < 0.05), Copromorpha (P < 0.05), and Paraprevotella (P < 0.05) were observably higher in DFT animals (Fig. 6a,b).

Fig. 6
Fig. 6
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Biomarkers among yak calves revealed by LEfSe. (a) Cladogram diagram, (b) Bar chart of LDA effect values for the indicator species.

The t-test showed that Firmicutes B 370539 was markedly higher in DFT than in DFO yaks (P < 0.05) (Fig. 7a). The abundance of Phocaeicola A 858004 (P < 0.05, P < 0.05), OLB9 (P < 0.05, P < 0.05), CAG-41 (P < 0.05, P < 0.01), Cryptobacteroides (P < 0.01, P < 0.01), CAG-273 (P < 0.01, P < 0.01) and Evtepia (P < 0.05, P < 0.05) in DFC yaks was markedly lower than them in DFO and DFT calves, respectively. In contrast, Faecalimonas (P < 0.05, P < 0.05) was signally higher in DFC yaks. The abundance of Choladousia (P < 0.05) and UMGS1071 (P < 0.05) was markedly higher in DFC animals than in CFO animals. Paramuribaculum (P < 0.05), CAG-488 (P < 0.01) and UBA3789 (P < 0.05) were significantly higher in DFO yak than in DFT animals. UBA5905 (P < 0.05, P < 0.05), UBA4334 (P < 0.05, P < 0.05) and WQUU01 (P < 0.05, P < 0.05) were memorably higher in DFO ruminants than DFC and DFT yaks. UBA737 (P < 0.05), CAG-269 (P < 0.05), Ructibacterium (P < 0.05), UBA2658 (P < 0.05), Agathobacter 164119 (P < 0.01), QAKW01 (P < 0.05) and Merdisoma (P < 0.05) were signally higher in DFO group than them in DFT group, while Limiplasma (P < 0.05), Peptococcus (P < 0.05), Bulleidia (P < 0.01), UBA9715 (P < 0.05), UBA11471 (P < 0.01) and Saccharofermentans (P < 0.05) were obviously lower in DFO animals. CAG-83 (P < 0.01), SFLA01 (P < 0.01), CCUG-7971 (P < 0.05), RF16 (P < 0.05), and Enterousia (P < 0.05) were markedly lower in the DFC group than in the DFT group. In contrast, RUG420 (P < 0.05) and Butyricicoccus A 77030 (P < 0.05) were observably higher in the DFC group. The abundance of Anaerobutyricum (P < 0.05, P < 0.05) was obviously lower in yaks in DFT than in animals in DFC and DFT (Fig. 7b).

Fig. 7
Fig. 7
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Biomarkers among yak calves are revealed by T- test. Data are presented as SD, *P < 0.05, **P < 0.01, ***P < 0.001, and ***P < 0.0001.

Discussion

Yaks are economic and religious farm ruminants on the plateau area of China, high-efficient breeding is of utmost importance to local people. Low reproductive performance is a long-time restrictive factor in the development of yak industry, and the long period of yak calves’ lactation is an important problem. Here in this study, we explored the effect of starter feeds on early weaning Chawula yak calves to explore novel highly efficient breeding methods in plateau regions.

Starter feeds promoted the growth of Chawula yak clave with observably higher weight (P < 0.05) and net weight growth rate (P < 0.001), especially in DFT animals (Fig. 1a), which was in accordance with previous studies7,19. Further study indicated that starter feeds enhanced the growth of chest girth in calves in groups DFO (P < 0.05) and DFT (P < 0.05) (Fig. 1b). T-AOC, SOD, MDA, and GSH-Px are four core enzyme markers indicating oxidation resistance and oxidative stress state of animals20,21. The elevated levels of T-AOC and GSH-Px in starter feeds fed ruminants, especially in calves in DFT groups (Fig. 1c), demonstrated that starter feeds could enhance the antioxidant ability of plateau animals. The improved growth performance observed in DFO and especially DFT calves was closely associated with enhanced antioxidant capacity, as evidenced by higher serum T-AOC and GSH-Px levels, suggesting a reduced oxidative burden that favors nutrient utilization and tissue accretion. Enhanced antioxidant status can support intestinal and ruminal homeostasis, thereby creating a more favorable environment for microbial fermentation and energy harvest. Antioxidant improvement in ruminants is directly linked to positive changes in their gut microbiota, particularly an increase in butyrate-producing microorganisms. Dietary antioxidants modulate the redox balance in the gut, creating a favorable environment that promotes the growth and diversity of beneficial, short-chain fatty acid (SCFA)-producing bacteria such as Lachnospiraceae and Ruminococcaceae families22. The increased abundance of these specific microorganisms leads to higher concentrations of butyrate, which serves as a primary energy source for colonocytes and plays a critical role in enhancing the ruminant’s own antioxidant capacity23. This synergistic interaction reduces overall systemic inflammation and oxidative stress, leading to a stronger intestinal barrier, improved nutrient absorption, and better growth performance in ruminants like lambs and calves.

High-throughput sequencing of the microbiota of yak calves in different groups and achieved 391520, 356907, and 353763 filtered sequences in DFC, DFO, and DFT yaks, respectively (Table 1). At the phyla level, the Firmicutes / Bacteroidota value in different groups was 0.40, 0.45, and 0.45 in DTC, DFO, and DFT yaks, which showed slight changes in starter feeds fed yaks, which may indicate the microbiota changes in yaks24,25. Then further we explored biomarkers among different Chawula yak calf groups and detected one phylum and thirty-seven different genera in different yak groups (Fig. 7). Among them, higher abundance of Phocaeicola A 858004, Cryptobacteroides and Evtepia was previous reported in yaks with higher weight26, healthy mice compared with animals with arthritis27, the higher abundance of those genus in yak calves in DFO and DFT groups may indicate that they are associated with the growth of yak in the plateau. Previous studies found that Faecalimonas was associated with skatole formation in pigs28, inflammatory response in colitis mice29. The lower abundance of this genus in supplemented yaks may illustrate that starter feed could inhibit the growth of this negative bacterium. Higher abundance of CAG-83 was found in yaks with higher weights26, and RF16 in pigs with good growth performance30, the enrichment of those two genus in DFT yaks may related with the growth promoting effect of starter feed. Anaerobutyricum is positively associated with the butyrate generation31, and butyrate is commonly known to have a favorable function on the homeostasis of the intestine and energy metabolism32. However, lower concentration of butyrate was found in lean people compared with obese humans33. Further study is needed to explore the relation between Anaerobutyricum and starter feed-treated yaks. Although overall rumen microbial diversity was not altered, distinct shifts in microbial composition and key biomarkers in DFT calves (e.g., enrichment of OLB9, Paraprevotella, and Akkermansia) indicate functional microbial reprogramming rather than diversity-driven effects. These microbial changes may enhance short-chain fatty acid production, epithelial integrity, and redox balance, collectively contributing to improved growth rates and chest girth development. Together, the results suggest that improvements in antioxidant capacity and rumen microbial composition act synergistically to promote better performance in yak calves.

Conclusion

We concluded that starter feeds could promote the growth of early weaning Chawula yak calves by enhancing antioxidant capacity and regulating the gut microbiota. One phylum (Firmicutes B 370539) and thirty-seven genera (Phocaeicola A 858004, Cryptobacteroides, Evtepia, CAG-273, etc.) were identified as biomarkers in yak calves. Our results may contribute to enhance the breeding efficiency of yaks in the plateau regions.

Materials and methods

Ethics approval

All procedures performed in this research were approved by the Laboratory Animal Welfare and Ethics Committee of Xizang Agricultural and Animal Husbandry University, and Nanjing Agricultural University (NJAU.No20240910164). This study is performed in accordance with relevant guidelines and regulations.

Animal experiment design

Twenty-one male yak calves (3 months) with near weight (22.05 ± 3.05 kg) from a local farm in Nierong County, China, were selected and divided into control group (DFC, n = 7), starter feed group 1 (DFO, n = 7), and starter feed group 2 (DFT, n = 7). Calves in the control group were free-ranged (DFC), the second group were fed with 1.4 kg/head/day alfalfa and 1.4 kg/head/day starter feed 1 (DFO), and third group was fed with 1.4 kg/head/day alfalfa and  1.4 kg/head/day starter feed 2 (DFT) for six weeks. The starter feeds were designed (starter feed 1 containing whey powder (5%), soybean flour (7.5%), fish powder (2.5%), expanded corn (20%), limestone powder (2.5%), calcium hydrogen phosphate (2.5%), salt (0.5%), vitamins (1.5%), additives (8%), and alfalfa (50%), and starter feed 2 containing whey powder (2.5%), soybean flour (3.75%), fish powder (1.25%), expanded corn (10%), limestone powder (1.25%), calcium hydrogen phosphate (1.25%), salt (0.25%), vitamins (0.75%), additives (4%), and alfalfa (75%)) and producted (Item No: 10175852138578) by Yingmeier Ltd., China. Calves’ weights and body sizes were measured, and blood samples were taken at three and six weeks of the experiment. At the end of the experiment, fresh fecal samples were collected and stored at − 80 °C.

Antioxidant capacity determination

All of the blood samples of yak calves were centrifuged to get serums for antioxidant capacity examination by employing commercial kits of T-AOC, MDA, SOD, and GSH-px (Nanjing Jiengcheng Bioengineering Research Institute Co., Ltd).

Fecal microbiota sequence

The genomic DNA of calf fecal samples was extracted by employing PureLink™ Microbial DNA Purification Kit (Invitrogen, USA). The quantity and quality of extracted DNAs of yak calves were examined utilizing NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, USA) and 1.5% agarose gel electrophoresis34,35. The valid DNA products of calves were employed for 16S rRNA (V3-V4) gene amplification via 338F/806R primer pairs36, and then those PCR generations were purified and quantified by piloting TIANgel Purification Kit (Tiangen, China) and Quant-iT PicoGreen dsDNA assay (Invitrogen, USA). Finally, amplicons of yaks were sent to pair-end 2 250 bp sequencing utilizing the Illumina MiSeq platform at Bioyi Biotechnology Co., Ltd (Wuhan, China).

Microbiota bioinformatics analysis of yak calves

The generated raw reads were demultiplexed, filtered, denoised, merged, and chimera removed to get quality sequences by employing DADA237. Non-singleton amplicon sequence variants were achieved by aligning yak sequences with MAFIT38. The ASVs’ taxonomy analysis of yaks was performed by aligning with the Green genes 2 database39, and a Venn diagram was drawn to visualize co-existing ASVs in different yak groups using the R package40. Then alpha (Chao1, Shannon, Simpson, etc.) and beta (Principal coordinate analysis, nonmetric multidimensional Scaling, etc.) diversities of yak calves were calculated via QIIME241. The difference in yaks among different groups was evaluated using PERMANOVA via QIIME242. The biomarkers of yaks in different groups were detected via methods of Linear discriminant analysis, effect size, and T-test43,44.

Statistical analysis

All of the results of yak calves in starter feed groups were compared with control group by performing student’s T-test via SPSS (27.0) to explore the effect of starter feed on animals. Data are presented as means ± SD, and statistical significance is considered when P < 0.05.