Effects of rumen-protected fat on changes of metabolites and reproductive genes in testes of Malin rams

The effects of RPF on metabolites and reproductive genes in testes of Malin ram was investigated. Twenty Malin rams (36.6kg ± 5.57 kg of bodyweight), were subjected to four dietary treatments; A: basal diet without rumen-protected fat (RPF), B; basal diet with 2% prilled fat, C; basal diet with 2% calcium salt and D; basal diet with 2% canola oil. At the end of the experiment four out of five animals from each group were slaughtered. The testes were excised for metabolites and gene expression studies. The genes tested were associated to testes development and spermatogenesis (ODF1, SERPINA10, CatSper4, AdipoR2 and DAZL). Feeding RPF with calcium salt (Treatment C) has resulted in the up-regulation more than two folds in all reproductive genes. There were metabolites changes occurred between the groups and identified 44 important putative metabolites present in the testes. In conclusion, feeding of RPF to the animals as a source of energy has up-regulated the genes and identified the metabolites involve in the male reproductive tissues and activities.


INTRODUCTION
Animal feeding is one of the main factors that can alter the performance of male animals. In the reproduction activities, rams that are not allowed to have proper feeding and nutrition will impact the breeding performance such as undeveloped testes, low sperm production, and quality which in turn will lead to reduce the fertility of the animals (Lone et al., 2017;Singh et al., 2018). To overcome these limitations, the farm-ers need to provide the rams with proper feeds, especially during breeding seasons. Grains that were incorporated in the pellet are a common practice to overcome these issues. However, as the price of the grains is getting higher these days, it would affect the farmers in terms of their finances. Alternatively, the use of fats as feed supplements during breeding seasons will help to resolve these issues (Schoech et al., 2004).
Currently, there is a technology to produce insoluble fats from microbial fermentation and biohydrogenation which is called rumenprotected fats (RPF) (Behan et al., 2019 andDe Silva et al., 2020;). This helps the RPF to escape the rumen microbial fermentation, absorbed through the small intestine and then converted to a source of energy (Owens and Basalan, 2016). There have been a few studies on the effects of RPF on metabolites and gene expression in female animals (Behan et al., 2019;Chavda et al., 2022), however, the current study focused on metabolites changes and reproductive genes in the ram's testes.
The sperm fatty acid composition is characterized by very high proportions of omega-3 PUFAs (n−3), particularly Docosahexaenoic acid (DHA; 22:6 n − 3) (Hosseini et al., 2019;Ngcobo et al., 2021). The ratio of omega-6 (n−6) to n−3 PUFAs may play a significant role in several aspects of animal production and reproduction (Khoshniat et al., 2020;Ngcobo et al., 2021). Furthermore, there is substantial evidence that the lipid composition of the sperm membrane is a major determinant of motility, overall viability, cold sensitivity, fusion capacity of sperm, and lipid metabolism (Wysokińska and Szablicka, 2021). There have been several studies on the effect of RFP on metabolites in ewes, however, our research focused on metabolites in the ram's testes. RPF increased Malin ram reproduction performance by enhancing sperm quality (Ahmad et al., 2018;Manriquez et al., 2019).
The development of testis and spermatogenesis are frequently regulated by core genes (Du et al., 2021;Zhang et al., 2019). There are many genes associated with nutrition and reproduction Heng Yang et al., 2018), therefore the current study focused on five differential genes (DEGs) which were; Outer density fiber protein 1 (ODF1), SERPINA 10, Cat ion channel of Sperm4 (CatSper 4), AdipoR2 and DAZL. These genes were chosen because of their functions in testis development and spermatogenesis (Bai et al., 2017;Qu et al., 2019). Gene expression is a process that involves transcription, translation, and turnover of messenger Ribonucleic acids (mRNAs) and proteins (Naval-Sanchez et al., 2018). It is regarded as one of life's most fundamental processes, and the genes expressed in an organism define its characteristics or features (Buccitelli and Selbach, 2020). To improve animal reproductive performance, many farmers now use rumen-protected fat. RPF improved Malin ram reproduction performance by improving sperm quality (Ahmad et al., 2018), and the current work was a continuation of that research to further understand RPF effects at the molecular level. Therefore, this study aimed to investigate how rumen-protected fat altered metabolite changes, and mRNA expression of reproductive genes involved in testes development and spermatogenesis in Malin ram testes.

Experimental Design, Animals and Diets
Twenty Malin rams (BW of 36.6 kg ± 5.6 kg at 10-14 months of age, with body scores 3.0 -3.5) were reared at the National Institute Animal Biodiversity Jerantut, Pahang and approved by the Universiti Putra Malaysia animal care and use committee (IACUC) guidelines (Reference # R064/2016). The experimental animals were divided randomly into four treatment groups, each of which received a different feeding treatment: A: basal diet without rumen-protected fat (RPF); B: a basal diet with RPF (as 2% prilled fat from palm oil source); C: basal diet with 2% RPF (as calcium soap, Casa); and D: basal diet with 2% canola oil (Table 1). The 12-week experiment included a two-week adaptation period and ten weeks of feeding trials. Each group received isocaloric and isonitrogenous formulated feed according to the maintenance requirement of sheep with water provided ad-libitum. The animals were dewormed with Fenbendazole 10%, 1ml/10kg two weeks before the feeding trials.

Testes Collection
Four Malin rams' testes (four from each treatment group) were taken as described in our earlier work (Ahmad et al., 2018) to determine metabolites and gene expression in testes. The testes samples were stored at -80 °C for further gene expression study.

Sample Preparation and LC-MS Analysis
The testes' tissues were crushed into a fine powder using a pestle and mortar with liquid nitrogen. The testes tissue samples were prepared as described in previous studies (Chen et al., 2015;Fraser et al., 2021), with slight modifications. Briefly, approximately 150 mg of each pulverized sample was homogenized in 450 μl methanol in two steps to make a final volume of 900 μl to precipitate the proteins. The solution was vortexed three times for five minutes each, then centrifuged at 16000 x g for 15 minutes at 4 °C. A centrifugal evaporator was used to concentrate the sample to a final volume of 75 μl. The sample was then centrifuged at 17,500 × g for 5 min at 4 °C and the supernatant was transferred to a new 1.5 ml microcentrifuge tube for LC-MS analysis. Each biological sample was replicated four times.
Fingerprinting sample was performed using Agilent 1200 LC system with an auto-sampler and binary pump coupled to 4000 Q-TRAP (AB Sciex, USA). Chromatographic separation was performed on zorbax eclipse xdb -C18 150 x 4.6 mm x 5 u. Mobile phases consisted of solution A; 0.1% formic acid in ultrapure water and solution B; 100% acetonitrile with the following gradient conditions; 0-1 minute 97% of solution A and 3% of solution B. From minutes 1 to 19 minutes, solution B was from 3% to 97% of concentration. Then it was maintained until 22 minutes. The column was equilibrated before each analysis. The flow rate was 0.5ml/min and the volume of the sample injected was 15μl. Analysis was performed at 45°C. The mass spectrometer was operated in a positive mode in scan type of Enhance MS (EMS). The scan rate was 1000Da/s and the scan range of 50-2800 m/z.

Total RNA Extraction and Purification
Total RNA was extracted from sample tissues using the RNeasy® Lipid Tissue Mini kit (Cat. No. 748sw04,Qiagen,Hilden,Germany) according to the manufacturer's protocol. Next, using a spectrophotometer (NANODROP (ND 1000), 1 µl of RNA was used to evaluate the purity of RNA by measuring the absorbance ratio at 260 nm/280 nm, and only samples with a ratio of ̴ 2.0 were generally accepted as "pure". Furthermore, the purification of RNA was determined by the evaluation of cDNA using standard curves.

Quantitative Real-Time Polymerase Chain Reaction (q-PCR)
The synthesis of first-strand cDNA was run by reverse transcription of 3 µg isolated total RNA (20 µl reaction mixture) as per kit protocol. The reaction was placed in the thermal cycler MJ Research PTC-100, USA. The master mix was prepared as per the manufacturer's protocols. The master mix was prepared according to the manufacturer's protocol. The 2x QuantiNova SYBR Green PCR Master Mix, QuantiNova Yellow Template Dilution Buffer, cDNA, primers, and RNase-Free water were thawed. The primers sequences of the selected gene which were forward and reverse are presented in Table 2. Each reaction (20 μL) contained 10 μL 2x SYBR PCR Master Mix, 1 μL of each forward and re-verse primers, 7 μL of nuclease-free water, and 1 μL of cDNA. The qPCR reactions were carried out following standard cycling mode as per kit protocol. A melting curve was also generated to confirm the sequence-specific PCR products. Two house-keeping genes of Glyceraldehyde 3phosphate dehydrogenase (GAPDH) and Betaactin (β-actin) were used in triplicates to determine the stable house-keeping gene in tissues. Real-time PCR was then performed on a Bio-Rad CFX Manager™ 3.1 real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA). Genes of interest were amplified through the 3 steps cycling program; step 1; denaturation for 15 seconds at 95°C; step 2; annealing for 20 seconds at 60°C; and step 3, extension for 20 seconds at 72°C. A standard curve was constructed to determine if the sample can be either a genespecific plasmid or a cDNA preparation in which the gene of interest is present. The R 2 of the standard curve should be > 0.980 (Qiagen, Germany). For the efficiency, reproducibility, and dynamic range of an SYBR Green assay, the standard curve was constructed using serial dilutions of 10 -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5 . The efficiency of the assay should be 90-105%. Quantification analysis was performed by measuring the average cycle threshold (CT) value ∆∆ (2-∆∆CT method) described by Livak and Schmittgen, (2001) using the formula below, following the standard curve method after normalization with reference genes.
In the above equation, Etarget and Eref are the amplification efficiencies of the target and references genes, respectively. ∆CT target (calibrator-test) = CT of the target gene in the calibrator minus the CT of the target gene in the test sample and ∆CT, ref (calibrator -test) is the CT of the reference gene in the calibrator minus the CT of the reference gene in the test sample.

Data Processing
The success of the LC-MS technique can be determined by its ability to give three dimensional (3D) data. First, the compounds were separated in time by LC (retention time). Secondly, the ions generated in the ionization source were then separated according to their mass to charge ratio, and m/z ratios in the mass analyser of MS, and finally, the MS detector measured the abundance of each ion (intensify). For data processing, the ion sources data then went to further screening, verify and quantitate using the Analyst 1.5.1 software. During the pre-screening, the outlier ions were removed from the data. The cleaned data were then reformed into an excel matrix for further analysis.

Data Analysis
The excel matrix was exported to SIMCA-P software (version 14.1, MKS Umetrics, Sweden) for further multivariate statistical analysis. The data were employed to identify biochemical patterns using principal component analysis (PCA), partial least squares-discriminate analysis (PLS-DA) and orthogonal partial least squares discriminate analysis (OPLS-DA). Some outlier ions also need to be removed along the process to get the most relevant ions. The values of variables' importance in the projection (VIP) in the OPLS-DA model were used to determine important metabolites. The characteristic of metabolites (the exact molecular mass and m/z) were identified using the Human Metabolome Database (http://hmdb.ca/spectra/ms/search), and the MassBank database (www.massbank.jp/search), Metabolites and Tandem MS Database (http:// metlin.scripps.edu) and the RIKEN integrated database of mammals (http://scinets.org/db/ mammal).

Statistical Analysis
Data were analyzed using Statistical Analysis Software (SAS, version 9.4). General Linear Model (GLM) was used to analyse the data and Duncan's Multiple Range Test (DMRT) was used to compare the mean between the treatments. Data were expressed as (mean ± SE) and statistical analysis that has a value of P < 0.05 was considered significantly different (one-way ANOVA).

PCA, PLS-DA and OPLS-DA of Metabolomics Profile
PCA, an unsupervised pattern recognition method, was used to determine the presence of inherent similarities in spectral profiles. Each scatters represented the testes sample in every treatment group. The PCA and PLS-DA result showed that there were not any separation between the groups or overlapping each other. To differentiate the testicular metabolites, we applied the OPLS-DA model to characterize the control and each different dietary group. There was a distinct clustering between the control group and each treatment group. However, the clustering between group C and group D was not well separated (Figure 1; OPLS-DA). The completely separated clustering between the treatment groups indicated that control group A could    be attributed to have different metabolites from groups B, C, and D. The variable importance for the projection (VIP) summarizes the importance of the variables to explain and correlate to Y. The plot was sorted from high to low, to show the confidence intervals for the VIP value. VIP values were arger than 0.5 indicated "important" metabolites and lower indicated "unimportant" (Figure 2). Using VIP > 0.5 as the cut-off, the important metabolites were identified in the testes and listed in Table 3.
The corresponding Loading's scattered (LS) plot showed the distribution of different variables between control and treatment groups. Each point in the LS plot represents an ion. Ions far away from the origin are significantly important to the differences between groups. Ions in Figure  3, were illustrated and given the identification according to their groups as shown in Table 4.
In the control group (A), 10 -hydroxy -(2E,8E) -decadiene -4 -ynoic acid was mainly identified. We found the metabolite in the group VIP are Adenosine 5' pentaphosphate and PE (24:0/24:1(15Z)). Adenosine is a product of complete dephosphorylation of adenine nucleotides which are presence in various compartments of the cell. PE (24:0/24:1(15Z)) is a phosphatidylethanolamine (PE or GPEtn). Fatty acids containing 16, 18 and 20 carbons are the most common, however PE (24:0/24:1(15Z)), in particular, consists of one chain of lignoceric acid at the C-1 position and one chain of nervonic acid at the C-2 position. While most phospholipids have a saturated fatty acid on C-1 and an unsaturated fatty acid on C-2 of the glycerol backbone, the fatty acid distribution at the C-1 and C-2 positions of glycerol within phospholipids are continually in flux, owing to phospholipid degradation and the continuous phospholipid remodeling that occurs while these molecules are in membranes. PEs are neutral zwitterions at physiological pH. They mostly have palmitic or stearic acid on carbon 1 and a long chain of unsaturated fatty acid (e. g. 18:2, 20:4 and 22:6) on carbon 2 (PubChem, U.S. National Library of Medicine). The synthesis of Adenosine and PE could be due to the increment of the metabolism rate in the cells and the various process involved in the spermatogenesis in the testes in the animals' group fed with calcium salt and its efficiency to produce energy (Pavkovych et al., 2015). Basch et al. (1992) reported that a Ca2+and Mg2+ stimulated adenosine 5' diphosphatase has been found in lactating bovine mammary glands. The enzyme is associated with membranes of mitochondrial, microsomal, and Golgi  Figure 3. Loading's scatter (LS) plot illustrated the metabolites that consist in different treatment groups apparatus fractions in the mammary gland which indicates a possible role for this enzyme in the milk secretory process, particularly in ATP cycling in vesicles. Therefore, Kurebayashi et al., (1980) mentioned that Adenosine 5' pentaphosphate represents an extremely useful tool in experiments with fragmented sarcoplasmic reticulum, such as studies of H + movement accompanying Ca + movement, ATP-ADP exchange reaction, and calorimetry of the Ca + uptake process in bullfrog skeletal muscle. Cusostomo et al., (2020) reported that the increase in testicular content in ATP and ATP/ ADP ratio was correlated with sperm counts. He also identified polar lipid metabolite in testicular of mice which are Glycerol, Ethonolamine, Phosphoethamolamine and Myoinositol using the H-NMR.
In general, the LIPID MAPS comprehensive classification system for lipids is comprised of eight lipid categories: Fatty acyls (FA), Glycerolipids (GL), Sphingolipids (SP), Glycerophospholipids (GP), Saccharolipids (SL), Prenol lipids (PR), Polyketides (PK), and Sterol lipids (ST). Each lipid category has its subclassification hierarchy. Glycerolipids (GL) are mono-, di-, and tri-substituted glycerol's, the most well-known being the TAGs, fatty acid esters of glycerol, formerly termed as triglycerides. TAGs represent the most abundant lipid class in oils and fats of animal origin and comprise the bulk of storage fat in mammalian tissue. These molecules exist as enantiomers since a centre of asymmetry is created upon enzymatic biosynthesis at carbon 2 of the glycerol backbone (Donato et al., 2015). From this finding, we could understand that the important putative metabolites in the testes were fatty acids and their derivatives. This is consistent with Jafaroghli et al. (2014) who mention that lipids are abundant in testicles, playing a crucial role in membrane structure and function, energy storage and cell signaling.

Reproductive Genes
The relative expression of the outer dense fiber protein 1 (ODF 1), SERPINA10, CatSper4, AdipoR2 and DAZL are presented in Figure 4. In the group fed with calcium salt and canola oil, the expression levels of ODF 1 were five-fold and seven-fold compared to the control. SERPINA10 has expressed up-regulation compared to control which is four-fold, eight-fold, and 15-fold in the group fed with prilled fat, calcium salt, and canola oil. CatSper4 was expressed up to four-fold in calcium salt meanwhile, in canola oil two-fold compared to control. The AdipoR2 gene was only expressed in the group fed with calcium salt which is ten-fold compared to the control. DAZL gene was expressed five-fold in both groups; the group was fed with calcium salt and canola oil, respectively.
Ram fertility is important and it is influenced by testis development and spermatogenesis. Both testis development and spermatogenesis are often regulated by core genes (Bai et al., 2017). Feeding RPF with calcium salt resulted in the up-regulation of all genes studied in this study (ODF1, SERPINA10, CatSper4, AdipoR2 and DAZL), with more than two fold increment. This is because calcium salts have the highest digestibility among the other unsaturated fatty acid sources, providing the highest digestible energetic value which leads to the up-regulation of these core genes (Block et al., 2006). ODF1 and Catsper4 are the genes related to sperm total number, concentration, and progressive motility (Ahmad et al., 2018). SERPINA10 gene can improve fertilization and sperm development (Bai et al., 2017). Lipid metabolism with PUFAs improves gene expression of AdipoR (Mazaherioun et al., 2017), and DAZL gene which will bring up the meiosis and spermatogenic process in the testes (Ma et al., 2013). The present findings are congruent with the report made by Pavkovych et al., (2015), where supplementation of protected fats and polyenoic fatty acids of vegetable origin in a diet of cattle stimulates metabolism in the animals, increases their productivity, and improves the quality of milk and meat. Supplements of calcium salts of fatty acids, made of palm oils, soybean, sunflower, rapeseed, and flaxseed are best given in a diet to young animals. Present finding suggested that supplemen-tation of RPF in the form of calcium salt may improve the fertility of Malin ram through sperm total number, concentration, and progressive motility.

CONCLUSION
It could be concluded that feeding lipid as RPF with calcium salt had converted PUFAs to Adenosine, or energy for the spermatogenesis process in the testes. The OPLS-DA model showed that there was a metabolites difference or changes between the control and RPF dietary group. However, no changes were identified between the calcium salt and canola oil. The changes indicated the efficiency of the RPF metabolism to produce PUFAs, TAG, PE, and Adenosine during the spermatogenesis in the testes. The present study also revealed 44 important putative metabolites via preliminary screening of LC/MS. However, this study only gives an overview of the metabolites in the testes and the changes when the animals are fed with prilled fat, calcium salt, and canola oil. The NMR or LC-MS/MS and Proteomic integration with pathway analysis should be performed to confirm the putative metabolites and pathways in this study. Similarly, feeding RPF with calcium salt gave up-regulation effects on reproductive gene expression such as ODF1, SERPINA10, CatSper4, AdipoR2, and DAZL in Malin ram. This finding gave the impression that supplementation of RPF with calcium salt will improve the reproductive efficiency in male ruminants by improving the motility of the sperm as well as the meiosis and spermatogenic process in testes.