Serum Amino Acids are Differentially Present in the Buffalo Estrous Cycle

Domestic animals, such as buffaloes, perform crucial functions in rural economy (Nakao and Singh-Nanda, 2003). Reproduction of animals is a crucial step in ensuring the continuation of their generation for the benefit of humanity (Narita et al., 2011). Estrus initiates the propagation of the next generation. This is influenced by several factors, including amino acids, which are essential for many physiological activities (Bhatia et al., 2020; Nayan et al., 2020, 2021; Onteru et al., 2016; Ravinder et al., 2016; Selvam et al., 2017; Surla et al., 2021). Cytosolic O-glycosylation of glutamine stimulates production of argininosuccinic synthetase gene (Brasse-Lagnel et al., 2003). Supplementing with L-arginine influences gene expression in adipose tissue (Jobgen et al., 2009), reproductive performance and metabolism (Elango et al., 2009; Mateo et al., 2007). Previous studies (Bronte and Zanovello, 2005) done on amino acids in many animals have opened the door to a hitherto unknown field of amino acids and their significance. When methionine has been added to the diet “instead of any other single amino acid, the inhibition of fecundity imposed on an amino acid-deficient diet has been alleviated. According to” this evidence, dietary quantities of amino acids, either in absolute or balanced amounts, might influence reproductive function in addition to caloric content (Bass et al., 2017).

According to research, “dietary amino acids additionally regulate transcriptional activity of hepatic estrogen receptors via mechanism that is dependent on mammalian target of rapamycin (mTOR). In mice, liver produces insulin-like growth factor1tr(IGF-1) in response to stimulation by hepatic estrogen receptor alpha”. This molecule signals reproductive system of the nutritional status of mice (della Torre et al., 2011). It is intriguing to consider if modifications in the diet’s amino acid composition would have an impact on female reproductive function since these hormone levels, especially IGF-1 levels, are strongly influenced by the diet’s protein and amino acid contents. Though many studies were done in different physiological conditions, there is a substantial research gap in amino acid composition during the estrous cycle in buffaloes. Because of the gaps existing, questions were asked, do amino acids differ from estrus and diestrus? If they differ what are the amino acids that were different in two stages? With these questions, it was hypothesized that there is a significant difference in amino acids during the estrous cycle.

Selection of buffalo heifers
 
During the winter (October to February 2020-21), 40 buffalo heifers from the Central Institute for Research on Buffaloes, Hisar, Haryana (Fig 1) had been selected by considering heat symptoms including vulval lip swelling, cervical secretions and restlessness. Hisar is 212 m above mean sea level, latitude is 29.17 N and longitude is 75.72 E. These were studied further as follows.


 
Estrus confirmation  by ultrasonography
 
While being held in a trevis, the selected heifers’ vulval lips were disinfected and washed following aseptic techniques. A Hitachi transrectal probe with a frequency of 7.5 MHz then inserted into rectum and gently moved across the uterine surface for examining the follicles and ovaries. Heifers having follicle diameters more than 12mm have been determined as estrus animals and follicular length has been determined by examining ovary impressions.
 
Estrus confirmation by salivary fern pattern
 
A sterile Pasteur pipette has been employed for collecting saliva that had been deposited on the lower lip in a micro-centrifuge tube prior to the animals being fed. Saliva samples had been collected at 6:30 a.m. every morning. 40 samples of saliva have been collected on average, once daily. For removing any feed, soil, or cells,  saliva has brought to lab on ice during oestrus period and centrifuged for 10 min at 4^oC at 3000 Xg. They transferred the supernatant to another container. A smear has been prepared on sterile glass slide utilizing 20 μl of cell and dirt-free saliva. (Ravinder et al., 2016). Before being inspected for fern pattern or salivary crystallization employing an inverted microscope (Olympus, Magnus INVI, Japan), this had been exposed to air for 5-10 min. They were subsequently later captured as photographs.
 
Blood collection
 
In clot activators, blood (10 ml) have been collected from screened and selected buffalo heifers exhibiting estrus symptoms by aseptically puncturing left jugular vein. To extract serum, collected blood has been left at room temperature for 4hr. Serum has been extracted, centrifuged for 8 min at 2000 g, then aliquoted in 2 ml falcon tubes for additional processing.
 
LC/MS/MS sample preparation
 
In 1.5 mL polypropylene microfuge tube, 100mL of serum has been combined with 850mL of methanol and 50 mL of internal standard. Exception of "D3-MET and D2-tyrosine (TYR)", that have been detected at 10 and 25 mmol/L, accordingly, “internal standards remained present at a final concentration of 50 mmol/L. 400 mL of” methanolic extract had been thoroughly dried under nitrogen when mixture has been vortexed, permitted to stand for 10 min, then centrifuged for 3 min at 10,000 g. The sample residue had been mixed with 100 mL of 3NHCl/butanol and incubated in capped borosilicate vial at 60^oC for 7.5 min. Upon mutilation, mixture had been reconstituted in 250 mL “of mobile phase (20% acetonitrile, 0.1%formic acid), dried entirely under nitrogen, then shifted to borosilicate autosampler vial for injection". Analysis of "LC/MS/MS" "An API 3000 triple quadrupole mass spectrometry equipment" "(Applied Biosystems, Foster City, CA, USA)” has been employed for executing MS/MS analysis. Flow has been split 1:4 into collision gas at 10 mTor, nebulizer gas at 14 mTor, curtain gas at 12 mTor and an "electrospray ionization(ESI)" source running at 325^oC in positive mode at 2000V. Multiple Reaction Monitoring (MRM) mode has been employed for collecting data utilizing "Analyst software (version 1.4.2)”. Each amino acid’s optimum fragmentation patterns had been determined at concentration of about 10mM. Polypropylene glycol has been employed for adjusting MS to unit mass resolution (±0.7 AMU). An HPLC system (1100) (Waters, Germany, CA, USA) has been employed to introduce 10 mL of sample. Samples have been added to an isocratic flow of 0.1% formic acid and 20% acetonitrile at rate of 1ml/min. A C 8, 4.6 12.5 mm 5 mm guard column and a 4.6 150 mm 5 mm resolving column (Zorbax Eclipse XDB-C8, Agilent) had been utilized for chromatography. There had been a 22-minute interval between injections. When 1000mmol/L sample had been examined in triplicates, carryover had been determined employing sample blank and had been 1 μmol/L for all amino acids.
 
Statistical analysis
 
Graphpad Prism version 9 has been employed for conducting a paired t-test at different significance levels (P<0.001 and P<0.05).

Ultrasonography revealed presence of 12–15 mm follicles, confirming that the animals were in the estrus stage (Fig 2). Additionally, a distinct salivary fern pattern, a clear indicator of estrus, was observed (Fig 3). Results have been consistent with observations reported by Bhat et al., (2014). The follicle measured 15 mm on ultrasonography (Morris and Billiar, 1994) and revealed a substantial salivary fern pattern (Zhang et al., 2017).



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Amino acids
 
Many amino acids and their intermediates were found to be markedly differentially expressed out of all amino acids, arginine, asparagine, β-alanine, cysteine, glutamine, histidine, gamma-hydroxylysine, hydroxyproline, isoleucine, leucine, tryptophan and valine have significantly differed at P<0.001 and at P< 0.05 as given in (Fig 4,5,6; Table 1) with diestrus group. However, argininosuccinic acid, aspartic acid, beta-amino isobutyric acid, glutamic acid, glycine, lysine, methionine, N-methyl histidine, threonine, proline, tyrosine, methyl histidine, had been insignificant (Fig 4,7; Table 2).

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The levels of L- alanine were observed more during estrus than diestrus. These findings are in agreement with Gatien et al (2019) and suggest L-alanine plays a crucial role in glucose metabolism. Low levels of L-alanine concentration during diestrus hypothecates the L-alanine involvement in higher production of glucose through gluconeogenesis. Seli et al., (2014) observed that in oocytes L-alanine had a role in cholesterol synthesis in the presence of growth factors (Fig 8). However, since there were no conclusive studies on glucose levels during estrus and diestrus, it is difficult to conclude the role of L-alanine in glucose metabolism. Arginine levels are elevated during the diestrus phase compared to the estrus phase, consistent with previous findings (Gatien et al., 2019), indicating that these variations may be driven by hormonal fluctuations. In several species, arginine has been demonstrated for improving specific reproductive processes, that includes placental and fetal growth (Redel et al., 2016). Besides, arginine has affected ovarian functions in several cells, that includes granulosa and luteal cell secretory activities, follicular survival and/or luteal cell apoptosis (Jain et al., 2012), an increase in arginine concentration during the diestrus phase, in turn, catalyzes CYR61, a potential molecular mediator of angiogenesis in the CL (Fig 8). Its function in angiogenesis that follows luteal development is confirmed by higher levels of expression of CYR61 in developing CL. When combined, these results indicate that CYR61 may serve an essential function in controlling angiogenic switch in Corpus luteum’s lifespan (Van Winkle et al., 1990).



The most prevalent amino acid had been glycine, that is conditionally required. However, since it is a necessary precursor for the production of proteins and nucleic acids, glycine is known for having an essential function in the early stages of embryonic development. Additionally, glycine is necessary for cells to proliferate rapidly (Downing et al., 1995). Additionally, glycine controls intracellular pH and could protect preimplantation embryos from osmotic stress (Grussing et al., 2016).  Transporter of glycine GLYT1 is a protein that transports glycine into cells and regulates cell volume. It is activated during oocyte maturation (Baltz and Tartia, 2009).

Leucine levels are higher during the estrus period as it is essential during the conversion of the preantral follicle to the antral follicle (Orsi et al., 2005). Additionally, recent research demonstrated that glycine and cysteine transport levels increased during oocyte maturation (Collado-Fernandez et al., 2012).

Elevated levels of histidine during estrus day prepare the uterus for preimplantation (Grussing et al., 2016). Hormonally controlled histamine synthesis is considered to assist in preparing the uterus for embryo implantation (Wood et al., 2000). HDC is the rate-limiting enzyme in histamine production. Preimplantation mouse uterus expressed large quantities of HDC mRNA, increasing on day four. This occurred around the same period of the estrous cycle as when lupin grain or glucose infusion increased rate of ovulation. Injecting same amino acids into ewes increased ovulation by 20% (Miller et al., 1986). On estrus day levels of leucine and valine are more than diestrus. Leucine and valine enhance ovulation rate by balancing negative energy balance throughout estrus cycle (Grussing et al., 2016). Leucine interacts with tissue synthesis pathways including IGF-1 and mTOR, which could assist with preovulatory follicles and luteal tissue proliferation. A slight increase in dietary leucine impacts prolactin synthesis and release, enhancing estrus cycle progression.

All metabolic activities in body depend on amino acids. Their function in estrous cycle regulation has been demonstrated in the current research. Significant differences were observed in alanine, arginine, anserine, histidine, tryptophan and isoleucine levels between estrus and diestrus stages (P<0.001). Significant differences in cystine and glutathione levels have been observed between stages (P<0.05). Contrary to these insignificant differences were noted for other amino acids across the two stages. These amino acids may regulate estrus cycle, according to these results. However, an in-depth understanding of amino acids in buffaloes remains a distant goal. A comprehensive study is required in the future to elucidate the roles of amino acids and the pathways they influence.

Current research has been supported by BMGF, CABin project and CIRB.
 
Disclaimers
 
The authors’ views and conclusions don’t always reflect those ²of their affiliated institutions. Although the authors take responsibility for the quality and accuracy of the information they provide they disclaim all liability for any losses, whether direct or indirect, that might result from² utilizing this content.
 
Informed consent
 
All animal procedures for experiments have been approved by Institute Animal Ethics Committee² of CIRB vide IAEC-CIRB/19-20/A/016 dated 05.08.19.

The authors of this article state that they have no conflicts of interest about its publication. No funding or sponsorship influenced design of research, data collection, analysis, decision in publishing, or preparation of manuscript.