Follicle stimulating hormone increased after cows parturition (lasting 2 ~ 3 d). When the follicle grows to a certain stage, the difference between the growth rate of the largest follicle and the second largest follicle reaches the maximum, and follicle deviation occurs (Ginther. 2016). The maximum diameter of the follicle may decrease due to the decrease of luteinizing hormone (LH) pulse. When the progesterone (P4) level in the blood decreases, the number of LH pulses will increase, and the diameter of the dominant follicle will continue to grow (Cooperative Regional Research Project. 1996). However, the dominant follicles fail to ovulate due to inadequate LH production, which is the direct cause of IO. In addtiton, high-producing cows with subclinical ketosis had low cholesterol or high liver perfusion since high energy metabolic demand, which may reduce steroid hormones synthesis or increases the clearance rate of ovarian steroid hormones, and then leads to anovulation and persistent corpus luteum (Petersson et al. 2006). The consumption of excessive lactation in high-yielding dairy cows were unable to provide more energy for the reproductive system, it may hinder the growth and development of follicles (Butler et al. 1981).
In our study, the clinical estrus, follicular development, uterine involutionm, ovarian stiffness and dominant follicle diameter were examined at 50 and 55 days postpartum. Our results showed that cows with IO had an average less than 8mm diameter of follicles (7.33 ± 0.42 mm) and less than 3mmd growth rate (0.4 ± 0.17 mm/d) within 5 days, and the other cows showed normally estrus behaviors and dominant follicles diameter (13.67 ± 0.71 mm), growth rate (1.4 ± 0.21 mm/d). Therefore, the follicular fluid of IO cows are collected in the secondary follicle stage (Nelson et al. 2017).
By comparing the serum biochemical indicators of E cows and IO cows in this study, the serum BHBA and NEFA levels of IO cows are higher, while the glucose is lower. It suggests an obvious subclinical ketosis (SCK) feature (Newman et al. 2016; Ihsanullah et al. 2017). Studies by Chang et al (2019) have also confirmed that after cows developed SCK postpartum 14–21 days, nearly 50% of them showed IO during postpartum 60–90 days. Serum BHBA and NEFA can inhibit the survival and growth of sheep preantral follicles and their oocytes cultured in vitro (Nandi et al. 2017). Glucose, TC and BHBA affect the oocytes maturation ability in dairy cows (Ferreira et al. 2011).
Changes of Serum Metabolites in IO Cows
Amino acids are one of the important components of protein and milk fat. In some early reports, some amino acids may affect the milk quality and production performance of dairy cows (Giallongo et al. 2016; Lee et al. 2015). In this study, there were seven serum DMs directly involved in amino acid metabolism in IO cows. Previous studies have confirmed that glutamine and glutamate can be converted into each other and directly participate in the tricarboxylic cycle for gluconeogenesis (Ardawi & Newsholme, 1990). Guanidinoacetic acid can generate creatine (Ostojic, 2019), in the form of phosphocreatine and free creatine, which provides energy for the body, alpha-ketoisovaleric acid can be converted into valine and together with isoleucine, it can regulate the blood sugar level to provide energy for the body (Xu et al. 2016). This study found that urea circulation will also be affected by participating in the tricarboxylic cycle since affecting the formation of fumaric acid through amino acids such as arginine (Yoshimi et al. 2016), or involving in the synthesis of antioxidants and anti-inflammatory factors (Liang et al. 2018; Zhao et al. 2018). The specific mechanism of amino acid metabolism affects inactive ovaries through energy metabolism or inflammation were still unclear untill now.
The carbohydrates screened in this experiment mainly involve pentose and glucuronate interconversions. For providing energy, the pentose phosphate pathway can offer raw materials for the synthesis of other substances, such as nucleotides and amino acids (Luo et al. 2007). In addition, L-ribulose can generate ribitol and carry out riboflavin metabolism, which participates in the energy response of the respiratory chain and cell growth and metabolism (Pinto & Zempleni, 2016). The blood L-ribulose and D-xylose levels are therefore reduced to maintain the level of pyruvate and some metabolic pathways such as the pentose phosphate pathway and riboflavin metabolism are weakened, which is not conducive to follicular development.
The study by Luo et al. (2019) showed that the content of phosphatidyl -choline in the serum of dairy cows increased during early lactation, which is consistent with these experimental results. Phosphatidylcholine homeostasis is particularly important for maintaining cell survival and growth. Studies have found that the total amount of phosphatidylcholine in cells is related to cell growth and apoptosis, positively related to growth and negatively related to apoptosis (Leng et al. 2018). Under the action of phospholipase, phosphatidylcholine can increase the production of arachidonic acid and linoleic acid (Seeley et al. 2006). The 13-HpODE produced by the oxidative metabolism of linoleic acid can enhance epidermal growth factor signal transduction by participating in the dephosphorylation of epidermal growth factor receptors (Hui et al. 1999) and inducing the expansion of the cumulus in the ovaries of mammals. Sphingomyelin can be metabolized to produce ceramide, which has important functions in barrier function, regulating cell function and participating in the signal transduction process which regulates cell growth and apoptosis (Hage et al. 2014). When culturing cells in vitro, adding ceramide can cause protein kinase inactivation and reduce the absorption of glucose by cells, and accelerate cell apoptosis (Hage et al. 2014). However, the relationship between ceramide and IO in dairy cows needs further study. In addition, this study found that 4-pyridoxine, a metabolite of vitamin B6, decreased in the serum of cows with IO. 4-Pyridoxic acid was formed from pyridoxal by aldehyde oxidase (AOX) in the liver. In previous studies among diabetic patients with vascular risk, 4-pyridoxine, and PAr [ratio of 4-pyridoxic acid/(pyridoxal + pyridoxal 5ʹ-phosphate)] index was higher in plasma and urine (Obeid et al. 2019), but its relationship with IO needs to be further explored.
Changes of FF Metabolites in IO Cows
In this study, the change trend of glutamine in FF and serum was consistent. The L-glutamine is the precursor and main energy source of nucleic acid biosynthesis, which enters the glycolysis and gluconeogenesis pathways or the purine and pyrimidine metabolism pathway. It can provide energy for the body (Summers et al. 2005), and promote the proliferation of cumulus cells in vitro (Sutton-McDowall et al. 2010). Excessive glutamine is transported to the cell through the transport system alanine-serine-cysteine (ASC), which may act as a competitive inhibitor of cysteine uptake. Cysteine is a key factor in the synthesis of glutathione in the g-glutamyl cycle (Yin et al. 2016), but it is still unclear whether glutamine in dairy cows with IO has an effect on the synthesis of glutathione, which in turn oxidizes and damages the follicular cells and hinders the development of the follicle. Other metabolites, such as L-valine, phenylpyruvic acid, and gentisic acid, can all participate in the tricarboxylic cycle, which in turn affects carbohydrates metabolism and lipid metabolism (Grochowska et al. 2001; Lemmon & Schlessinger, 2010).
The carbohydrates selected in FF are mainly related to starch and sucrose metabolism, pentose and glucuronate interconversions, and D-maltose as an intermediate substance that can convert glycogen into glucose. During the maturation of oocytes, both glucose and 6-hydroxy-5-methoxyindole glucuronide can provide energy for cells. In addition, glucose can also synthesize extracellular matrix substrates by cumulus expansion and O-linked glycosylation in cell signal transduction through the hexosamine biosynthesis pathway for follicular growth and regulate oocyte nuclear maturation and redox state through the pentose phosphate pathway (Cetica et al. 2002). Furthermore, 6-Hydroxy-5-methoxyindole glucuronide generates ascorbic acid which resists oxidation and scavenges free radicals (Chatterjee, 1978). In this study, the increase in maltose in the FF of IO cows and the decrease in 6-Hydroxy-5-methoxyindole glucuronide may enhance glycogenolysis.
Lipid-related metabolites mainly involve in glycerophospholipid metabolism and arachidonic acid metabolism. Phosphatidylinositol can be produced from serine and then enter the tricarboxylic cycle through acetyl-CoA or indirectly produce choline. It can also participate in the glycosylphosphatidylinositol (GPI) metabolic pathway, allowing cell membranes to bind to proteins (Jope et al. 1979; de Almeida et al. 2003). Studies have shown that choline can generate acetylcholine and enter the cAMP signaling pathway. Acetylcholine is a neurotransmitter, which not only affects the permeability of the membrane to ions, but also transmits signals through some second messengers and affects the physiological metabolic process. Studies have shown that in the ovaries cAMP is instrumental as a second messenger for the follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors (Conti, 2002; Zhang et al. 2004; Menon & Menon, 2012). The increased metabolism of glycerophospholipids may be related to signal transduction during follicular development of IO cows with SCK, but further research is needed. Arachidonic acid is elevated, which is consistent with the results of Moore S G (Wathes et al. 2007). Arachidonic acid participates in the ovarian production of steroid hormones through its metabolites, such as cyclooxygenase metabolism, to produce PGE2 and PGF2α, and lipoxygenase metabolism to produce leukotrienes. The PGE2 can promote the synthesis of hyaluronic acid and the expansion of the cumulus, or 5-HPETE produced by metabolism, which can affect the production of steroid hormones by regulating the expression of StAR protein (Eppig, 1981; Wang et al. 2003). However, Zhang (2019) pointed out that high concentrations of arachidonic acid can induce the death of granulosa cells in the ovary and inhibit the synthesis of estrogen by granulosa cells. This result may be because the catabolism of arachidonic acid is decreased and the content of FF is increased, which affects the synthesis of steroid hormones in granular cells.
Comparison of Metabolites in Serum and FF of IO Cows
In this experiment, UHPLC-QTOF-MS was used to analyze the serum and FF samples of the cows in the E and IO cows and 28 different metabolites were obtained. According to KEGG analysis of metabolic pathways, an interaction correlation diagram between DMs was constructed through integration in Fig. 3.
Scaramuzzi et al. (2011) pointed out that the SCK affects the growth of follicles induced at different levels of the hypothalamic-pituitary-ovarian axis (Scaramuzzi et al. 2011). Rodgers & Irving-Rodgers (2010) reviewed the regulation of follicular growth, fertility, and oocyte quality in ruminants. The FF from the blood needs to pass through the cortical interstitium, the basal layer of the follicle and the granular cell layer of the wall (Senbon et al. 2003). As the follicle develops, fluid will accumulate in the antral cavity of the follicle to provide nutrients for the development and maturation of the oocyte. The main function of follicles is to provide a blood to follicular barrier and create a favorable environment for growing oocytes. Similarly, oocytes play an important role in promoting the growth of follicles and directing the differentiation of granulosa cells, and interact with surrounding somatic cells such as granulosa cells (Senbon et al. 2003). In this study, L-glutamine and L-glutamate in the FF and serum were increased in dairy cows with IO, which was not conducive to improve follicular development. The increased L-glutamine in FF should derive from L-glutamate in the blood and then converted to pyruvate, which can provide energy for follicular development (Ardawi & Newsholme, 1990), mainly via alanine, aspartate and glutamate metabolism. In IO cows, the serum and FF phosphatidylcholine and its upstream and downstream metabolites were all elevated, mainly due to arachidonic acid metabolism. Although an appropriate concentration of arachidonic acid can participate in the production of steroid hormones, the high content of arachidonic acid in FF can induce granular cell death, which is not conducive to follicular development (Eppig, 1981; Wang et al. 2003). Thus, this study showed that sphingomyelin and lactosylceramide may be related to IO in dairy cows, but the specific mechanism needs further confirmation in the future.