The posttranscriptional gene regulation mediated by microRNA plays an important role in the development and function of male and female reproductive organs and germ cells in mammals, including cattle. In the present study, we identified novel and differentially expressed miRNAs in the testis and ovary in Holstein cattle by combining the Solexa sequencing with bioinformatics. In total 100 and 104 novel pre-miRNAs were identified in testicular and ovarian tissues, encoding 122 and 136 mature miRNAs, respectively. Of these, 6 miRNAs appear to be bovine-specific. A total of 246 known miRNAs were co-expressed in the testicular and ovarian tissues. Of the known miRNAs, twenty-one testis-specific and nine ovary-specific (1-23 reads) were found. Approximately 30.5% of the known bovine miRNAs in this study were found to have >2-fold differential expression within the two respective reproductive organ systems. The putative miRNA target genes of miRNAs were involved in pathways associated with reproductive physiology. Both known and novel tissue-specific miRNAs are expressed by Real-time quantitative PCR analysis in dairy cattle. This study expands the number of miRNAs known to be expressed in cattle. The patterns of miRNAs expression differed significantly between the bovine testicular and ovarian tissues, which provide important information on sex differences in miRNA expression. Diverse miRNAs may play an important regulatory role in the development of the reproductive organs in Holstein cattle.

Reproduction is vital for producing offspring and preserving genetic resources. However, incidences of many reproductive disorders (e.g. miscarriage, intrauterine growth restriction, premature delivery and lower sperm quality) have either increased dramatically or remained at high rates over the last decades. Mounting evidence shows a strong correlation between enteral protein nutrition and reproduction. Besides serving as major nutrients in the diet, amino acids (AA) are signaling molecules in the regulation of diverse physiological processes, ranging from spermatogenesis to oocyte fertilization and to embryo implantation. Notably, the numbers of bacteria in the intestine exceed the numbers of host cells by 10 times. Microbes in the small-intestinal lumen actively metabolize large amounts of dietary AA and, therefore, affect the entry of AA into the portal circulation for whole-body utilization. Changes in the composition and abundance of AA-metabolizing bacteria in the gut during pregnancy, as well as their translocation to the uterus, may alter uterine function and epigenetic modifications of maternal physiology and metabolism, which are crucial for pregnancy recognition and fetal development. Thus, the presence of the maternal gut microbiota and AA metabolites in the intrauterine environments (e.g. endometrium and placenta) and breast milk is likely a unique signature for the programming of the whole-body microbiome and metabolism in both the fetus and infant. Dietary intervention with functional AA, probiotics and prebiotics to alter the abundance and activity of intestinal bacteria may ameliorate or prevent the development of metabolic syndrome, while improving reproductive performance in both males and females as well as their offspring.

Thirty-six healthy piglets (weighing 10 ± 1 kg; three animals per pen) were randomly allocated to two treatments: (i) a low protein diet (14% crude protein [CP]) supplemented with lysine, methionine, threonine and tryptophan (Group LP) and (ii) a normal protein diet (20% CP, Group NP), resulting in six replicate pens per treatment. One piglet from each pen was slaughtered at days 10, 25 and 45 of the experiment. For the whole experimental period of 45 d, Group LP had lower feed intake and daily gain and a higher feed-to-gain ratio compared with Group NP. At day 10, no effects on measured caecum metabolites were observed, but at days 25 and 45 in Group LP the concentration of ammonia-N, cadaverine, branched chain fatty acids and acetate were reduced. This was also true for the concentration of short chain fatty acids at day 45. The results of denaturing gradient gel electrophoresis showed that microbial diversity in Group LP was less abundant at day 25, but there was no difference at days 10 and 45. An unweighted pair group mean average analysis showed that the similarities were lower between Groups LP and NP at day 10 and higher at days 25 and 45. Quantitation results indicated that the numbers of Firmicutes and Clostridium cluster IV were lower in Group LP than in Group NP at day 25, but there were no differences at days 10 and 45. In conclusion, the low protein diet markedly reduced the metabolites of protein and carbohydrate fermentation and altered microbial communities in the caecal digesta of piglets.

Major histocompatibility complex, class II, DQ alpha 2, also named BOLA-DQA2, belongs to the Bovine Leukocyte Antigen (BOLA) class II genes which are involved in the immune response. To explore the variability of the BOLA-DQA2 gene and resistance to mastitis in cows, the splice variants (SV), targeted microRNAs (miRNAs), and single nucleotide polymorphisms (SNPs) were identified in this study. A new SV (BOLA-DQA2-SV1) lacking part of exon 3 (195 bp) and two 3¢-untranslated regions (UTR) (52 bp + 167 bp) of the BOLA-DQA2 gene was found in the healthy and mastitis-infected mammary gland tissues. Four of 13 new SNPs and multiple nucleotide polymorphisms resulted in amino acid changes in the protein and SNP (c. + 1283 C > T) may affect the binding to the seed sequence of bta-miR-2318. Further, we detected the relative expressions of two BOLA-DQA2 transcripts and five candidated microRNAs binding to the 3¢-UTR of two transcripts in the mammary gland tissues in dairy cattle by using the quantitative real-time polymerase chain reaction. The result showed that expression of the BOLA-DQA2-SV1 mRNA was significantly upregulated 2.67-fold ( p < 0.05) in mastitis-infected mammary tissues (n = 5) compared with the healthy mammary gland mammary tissues (n = 5). Except for btamiR-1777a, miRNA expression (bta-miR-296, miR-2430, and miR-671) was upregulated 1.75 to 2.59-fold ( p < 0.05), whereas miR-2318 was downregulated in the mastitis cows. Our findings reveal that BOLA-DQA2-SV1 may play an important role in the mastitis resistance in dairy cattle. Whether the SNPs affect the structure of the BOLA-DQA2 gene or association with mastitis resistance is unknown and warrants further investigation.

Clone library of bacterial 16S rRNA gene was constructed to evaluate the bacterial diversity and community structure of uterus samples obtained from three postpartum healthy cows and three metritic cows on days 10 and 40. Sequences were assigned to five major groups (Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, and Tenericutes) and to an uncultured group. On day 10, Bacteroidetes, Firmicutes, and Fusobacteria were the dominant group both in healthy and metritic cows. On day 40, the major sequences were affiliated with Bacteroidetes, Firmicutes, Tenericutes, and Proteobacteria. Tenericutes (Ureaplasma diversum) were revealed only from healthy cows, while Proteobacteria (Histophilus somni) were found only from metritic cows. Quantitative PCR revealed that metritic cows on day 10 showed higher value of total bacteria, Bacteroidetes, Peptostreptococcus, and Fusobacterium compared with healthy cows, while only a higher value of Fusobacterium spp. was observed from the metritic cows on day 40 compared with that from healthy cows (P < 0.05). Our data indicates that great difference in the uterine bacterial community in both phyla level and species level exists between healthy and metritic postpartum cows, and dynamic changes in bacterial community occur over time.

The study aimed to determine the effects of reduction of dietary crude protein (CP) level with balanced essential amino acids (EAA) on intestinal bacteria and their metabolites of growing pigs. Forty pigs (initial BW 13.50 ± 0.50 kg, 45 ± 2 days of age) were randomly assigned to four dietary treatments containing CP levels at 20.00% (normal crude protein, NP); 17.16% (medium crude protein, MP); 15.30% (low crude protein, LP); and 13.90% (extremely low crude protein, ELP), respectively. Crystalline AAs were added to meet the EAA requirement of pigs. After 4-week feeding, eight pigs per treatment (n = 8) were randomly selected and slaughtered for sampling of ileal, cecal, and colonic digesta and mucosa. Pigs with moderately reduced CP level had increased bacterial diversity, with the Shannon diversity indices for the colon digesta in the LP group and mucosa in the MP and LP groups significantly (P < 0.05) higher than those in the NP and ELP groups. As the CP level reduces, the Bifidobacterium population were linearly decreased (P < 0.05) both in ileum, cecum, and colon, and the ELP group had the lowest Bifidobacterium population in the cecum and colon, with its value significantly lower than NP and MP groups (P < 0.05). However, the ELP group had the highest population of Escherichia coli in the colon, with its value significantly higher than the LP group (P < 0.05). For bacterial metabolites, as CP level decreased, total short-chain fatty acid (T-SCFA), acetate, and butyrate were linearly increased (linear, P < 0.05) in the ileum, while all SCFAs except formate in the cecum and T-SCFA and acetate in the colon, were linearly decreased (P < 0.05). Reducing CP level led to a linear decrease of microbial crude protein (MCP) in the ileum (P < 0.05) and ammonia in all intestine segments (P < 0.05). The spermidine in cecum and total amines, cadaverine, methylamine, and spermidine in colon were shown a quadratic change (P < 0.05) as dietary CP decreases, with the highest concentration in LP group. These findings suggest that moderate reduction of dietary CP level may benefit large intestinal bacterial community and its fermentation, which was negatively affected by extremely low CP diet.

High-mobility group box protein 1 (HMGB1) gene has a universal sentinel function for nucleic acid-mediated innate immune responses and acts as a pathogenic mediator in the inflammatory disease. In an effort to identify the functional single-nucleotide polymorphism (SNP) in the 3'-untranslated region (UTR) of the bovine HMGB1 gene that affects the binding to its target microRNA, first, the expression of HMGB1 mRNA in different genotypes and its candidate bta-miR-223 was investigated. Quantitative real-time polymerase chain reaction results showed that the relative expression of HMGB1 mRNA in cows with the genotype GG is significantly higher than those in cows with the genotype AA (P < 0.05). The expression of bta-miR-223 was significantly upregulated by 1.95-fold (P < 0.05) in the bovine mastitis-infected mammary gland tissues compared with that in the healthy tissues. Subsequently, luciferase assay indicated that the HMGB1 expression was directly targeted by bta-miR-223 in human embryo kidney 293 T (HEK 293T) cells. One novel SNP (g. +2776 A > G) in the HMGB1 3'-UTR, altering the binding of HMGB1 and bta-miR-223, was found to be associated with somatic count scores in cows. Taken together, the g. +2776 A > G-GG was an advantageous genotype which can be used as a candidate functional marker for mastitis resistance breeding program.

Plant flavonoids are generally regarded as natural replacers of synthetic growth promoters in poultry production. This study investigated the immunomodulatory effects of plant flavonoids, such as genistein and hesperidin, in lipopolysaccharide (LPS)-challenged broilers. A total of 700 21-day-old commercial Arbor Acres broiler chicks were randomly assigned into six treatment groups, each having six pens of 20 chicks/pen. Chicks were fed a basal diet without any additive (control, CON), 5 mg genistein/kg feed (G5), 20 mg hesperidin/kg (H20), or a basal diet with a combination of genistein and hesperidin (1 : 4) with doses of 5 mg/kg feed (GH5), 10 mg/kg (GH10) and 20 mg/kg (GH20) for 6 weeks. Half of the birds from each treatment were separated, and either challenged with 0·9% sodium chloride solution or Escherichia coli LPS (250 μg/kg BW) on days 16, 18 and 20. The results showed that both genistein and hesperidin improved (P<0.01) the plasma antioxidant status of growing broilers, by increasing total antioxidant capacity (TAOC), superoxide dismutase (SOD) activity and decreasing malondialdehyde production. LPS challenge further increased (P<0.05) TAOC and SOD levels. Regardless of LPS challenge, both genistein and hesperidin improved the humoral and mucosal immunity by increasing the intestinal intraepithelial lymphocyte numbers (P<0.01), as well as anti-Newcastle disease and anti-avian influenza antibody titers (P<0.05). Supplementation of both the plant flavonoids generally increased (P<0.05) the immune organs indices (spleen, thymus and bursa). Thus, supplementation of basal diet of broiler chicks, either with genistein or hesperidin, improved immune and antioxidant status of growing broilers. In addition, combined supplementation of both the flavonoids showed further improvement than individual compounds.