76 J. Indonesian Trop. Anim. Agric. 48(2):76-88, June 2023 

J I T A A 
Journal of the Indonesian Tropical Animal Agriculture  
Accredited by Ditjen Riset, Teknologi dan Pengabdian  

kepada Masyarakat No. 164/E/KPT/2021  

 

J. Indonesian Trop. Anim. Agric. 

pISSN 2087-8273 eISSN 2460-6278 

http://ejournal.undip.ac.id/index.php/jitaa 

48(2):76-88, June 2023 

DOI: 10.14710/jitaa.48.2. 76-88  

Effects of rumen-protected fat on changes of metabolites 

and reproductive genes in testes of Malin rams 

 
A. Ismail

1
, A. I.  Muhammad

1,2
, L. T. Kee

1
, L. T. Chwen

1
, and A. A. Samsudin

1*
 

1 
Department of Animal Science, Faculty of Agriculture,  

Universiti Putra Malaysia, Serdang 43400, Malaysia; 
2
 Department of Animal Science, Faculty of Agriculture, 

Federal University Dutse, Dutse 7156, Nigeria 

*Corresponding author: anjas@upm.edu.my 

  

Submitted February 14, 2023, Accepted March 28, 2023 

 

ABSTRACT 

 

 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 stud-

ies. 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 conclu-

sion, 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.  

 Keywords: Energy, Fat, Gene expression, Metabolites, Sheep. 

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 al-

lowed to have proper feeding and nutrition will 

impact the breeding performance such as unde-

veloped testes, low sperm production, and quali-

ty 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 

mailto:anjas@upm.edu.my


 

 

                                                               

Rumen protected fat and reproductive genes in Malin rams (A. Ismail et al.) 77 

biohydrogenation which is called rumen-

protected fats (RPF) (Behan et al., 2019 and De 

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 fe-

male 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 charac-

terized 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 as-

pects 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 ef-

fect of RFP on metabolites in ewes, however, our 

research focused on metabolites in the ram’s tes-

tes. RPF increased Malin ram reproduction per-

formance by enhancing sperm quality (Ahmad et 

al., 2018; Manriquez et al., 2019).  

 The development of testis and spermatogen-

esis are frequently regulated by core genes (Du 

et al., 2021; Zhang et al., 2019). There are many 

genes associated with nutrition and reproduction 

(Ma et al., 2019; Heng Yang et al., 2018), there-

fore 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 func-

tions 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 funda-

mental processes, and the genes expressed in an 

organism define its characteristics or features 

(Buccitelli and Selbach, 2020). To improve ani-

mal reproductive performance, many farmers 

now use rumen-protected fat. RPF improved Ma-

lin ram reproduction performance by improving 

sperm quality (Ahmad et al., 2018), and the cur-

rent work was a continuation of that research to fur-

ther 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 spermatogen-

esis in Malin ram testes. 

 

MATERIALS AND METHODS 

 

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 

Table 1: Feed ingredients of experimental diet 

Ingredients 
Experimental diets 

Control Prilled Casa Canola 

Brachiaria grass 66.00 76.00 75.00 77.00 

Commercial Sheep Concentrate 34.00 22.00 23.00 21.00 

Prilled Fat 00.00 02.00 00.00 00.00 

Calcium Salt of Fatty Acid 00.00 00.00 02.00 00.00 

Canola Oil 00.00 00.00 00.00 02.00 

Total (%) 100.00 100.00 100.00 100.00 

Calculated Analysis     

ME (Kcal/kg) 2217 2218 2210 2212 

Crude Protein (%) 12.20 11.30 11.30 11.20 

 

 



  

  78 J. Indonesian Trop. Anim. Agric. 48(2):76-88, June 2023 

use committee (IACUC) guidelines (Reference # 

R064/2016). The experimental animals were di-

vided 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 ani-

mals 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 pre-

pared as described in previous studies (Chen et 

al., 2015; Fraser et al., 2021), with slight modifi-

cations. 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 sam-

ple 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 anal-

ysis. 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, so-

lution 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 per-

formed at 45°C. The mass spectrometer was op-

erated 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 tis-

sues 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 pu-

rity 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”. Fur-

thermore, the purification of RNA was deter-

mined 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 manu-

facturer’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 se-

quences of the selected gene which were forward 

and reverse are presented in Table 2. Each reac-

tion (20 μL) contained 10 μL 2x SYBR PCR 



 

 

                                                               

Rumen protected fat and reproductive genes in Malin rams (A. Ismail et al.) 79 

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 pro-

tocol. A melting curve was also generated to 

confirm the sequence-specific PCR products. 

Two house-keeping genes of Glyceraldehyde 3-

phosphate dehydrogenase (GAPDH) and Beta-

actin (β-actin) were used in triplicates to deter-

mine 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 se-

conds at 60°C; and step 3, extension for 20 se-

conds at 72°C. A standard curve was constructed 

to determine if the sample can be either a gene-

specific 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, Ger-

many). For the efficiency, reproducibility, and 

dynamic range of an SYBR Green assay, the 

standard curve was constructed using serial dilu-

tions of 10
-1

, 10
-2

, 10
-3

, 10
-4

 and 10
-5

. The effi-

ciency of the assay should be 90-105%. Quantifi-

cation 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 dimen-

sional (3D) data. First, the compounds were sep-

arated 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 abun-

dance of each ion (intensify). For data pro-

cessing, the ion sources data then went to further 

screening, verify and quantitate using the Ana-

lyst 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 pat-

terns using principal component analysis (PCA), 

partial least squares- discriminate analysis (PLS-

DA) and orthogonal partial least squares dis-

criminate analysis (OPLS-DA). Some outlier 

ions also need to be removed along the process 

to get the most relevant ions. The values of vari-

ables' importance in the projection (VIP) in the 

OPLS-DA model were used to determine im-

portant metabolites. The characteristic of metab-

olites (the exact molecular mass and m/z) were 

identified using the Human Metabolome Data-

base (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 Analy-

sis 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 treat-

ments. Data were expressed as (mean ± SE) and 

statistical analysis that has a value of P < 0.05 

was considered significantly different (one-way 

http://hmdb.ca/spectra/ms/search)
http://hmdb.ca/spectra/ms/search)
http://www.massbank.jp/search
http://metlin.scripps.edu/
http://metlin.scripps.edu/
http://scinets.org/db/mammal)
http://scinets.org/db/mammal)
http://scinets.org/db/mammal)


  

  80 J. Indonesian Trop. Anim. Agric. 48(2):76-88, June 2023 

ANOVA). 

 

RESULTS AND DISCUSSION 

 

PCA, PLS-DA and OPLS-DA of Metabolom-

ics 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 be-

tween the groups or overlapping each other. To 

differentiate the testicular metabolites, we ap-

plied 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 com-

pletely separated clustering between the treat-

ment groups indicated that control group A could 

Table 2. Primer sequence of selected genes (Bai et al., 2017) 

Name of Target gene Primer sequence Product Size 

Cat ion channel of 

Sperm4 (CatSper4) 

F: TCGGCTGGTTAAATGGTTTC 114 

 R: CGACGGCACTGAGTTCATTA  

SERPINA10 F: TCTTACCCTGGGCTGACCTA 117 

 R: CTGCCATTGCCTCTGTACCT  

DAZL F: TTATCATGTGCAGCCACGTC 118 

 R: AGGGTTCATCATGGTTGGAG  

ADIPOR2 F: GAGGAGTGTGAGTGCGATGA 128 

 R: CGACCTTCCCAGACCTTACA  

Outer density fibre 

protein 1 (ODF1) 

F: CGCGAGAACAGATACGACTG 117 

 R: GAGCCCGTAGGAGTACGTCA  

β-actin F: GCTCTCTTCCAGCCTTCCTT 114 

 R: CGTGTTGGCGTAGAGGTCTT  

GADPH F: CATGGCCTTCCGTGTTCCTA 460 

 R: TACTTGGCAGGTTTCTCCAGG  

 

 

 

 

Figure 1. Multivariate analysis, OPLS-DA. A - control, B - prilled fat, C - calcium salt, and D - canola oil. 



 

 

                                                               

Rumen protected fat and reproductive genes in Malin rams (A. Ismail et al.) 81 

Table 3. Identified putative metabolites in the testes  

(Human metabolome database; http://hmdb.ca/spectra/ms/search, Mass Bank database; 

www.massbank.jp/search, Metabolites and Tandem MS database http://metlin.scripps.edu) 

No. VIP m/z Putative Metabolites Formula 

1 

 

1.89877 

 

102.0377 

 

10-hydroxy-(2E,8E)-decadien-4-ynoic Acid 

 

C10H12O3 
 

2 

 

1.82653 

 

102.0281 

 

albendazole S-oxide 

 

C12H15N3O3S 

 

3 

 

1.57053 

 

102.0238 

 

Nicotinuric acid 

 

C8H8N2O3 
 

4 

 

1.24246 

 

51.5657_11 

 

Methylcyclopentane 

 

C6H12 
 

5 

 

1.2031 

 

1528.0176 

 

CL(20:1(11Z)/18:2(9Z,12Z)/18:1(11Z)/18:1(11Z) 

 

C83H152O17P2 
 

6 

 

1.19827 

 

1293.3397 

 

18(R)-Hydroxy-20-oxo-20-CoA-LTE4 

 

C44H69N8O23P3S2  

 

7 

 

1.19754 

 

1680.3548 

 

CL(i-13:0/a-21:0/i-24:0/a-25:0)[rac] 

 

C92H180O17P2 

8 

 

1.17195 

 

116.0285_3 

 

Methylpentanoic acid 

1 Hexanoic acid 

 

C6H12O2 
C6H12O2 
 

9 

 

1.16402 

 

1401.0123 

 

DG(20:5(5Z,8Z,11Z,14Z,17Z)/ 

22:6(4Z,7Z,10Z,13Z,16Z,19Z)/0:0) 

 

C45H66O5 
 

10 

 

1.13656 

 

989.4541 

 

Fluspirilene 

 

C29H31F2N3O 

 

11 

 

1.11573 

 

1201.0583 

 

CE(12:0) 

 

C39H68O2 
 

12 

 

1.11306 

 

1121.1937 

 

Anhydrosafflor Yellow B 

 

C48H52O26 

 

13 

 

1.05437 

 

132.0922 

 

5,10-Pentadecadien-1-ol 

 

C15H28O 

 

14 

 

1.01708 

 

85.9785 

 

Crotonoic acid 

 

C4H10N2 
 

15 

 

1.01065 

 

58.8554_14 

 

1,1-Dimethylbiguanide 

 

C4H11N5 
 

16 

 

0.974863 

 

1051.2896 

 

Cyanidin 

 

C47H55O27 
 

17 

 

0.971319 

 

118.0185 

 

Succinic acid 

DL-2-Hydroxyvaleric acid 

 

C5H12NO2 

C5H10O3 

 

18 

 

0.97953 

 

104.0473 

 

DL-2,3-Diaminopropionic acid 

Choline 

3-Cyanopyridine 

 

C3H8N2O2 

C5H14NO 

C6H4N2 

 

19 

 

0.939066 

 

57.7020 

 

2-Amino-5-phenyl pyridine 

 

C11H10N2 
 

20 

 

0.936192 

 

85.9790 

 

Piperazine 

 

C4H10N2 
 

21 

 

0.934178 

 

1866.7223 

 

Glycerol trinonadecanoate 

 

C60H116O6 
 

22 

 

0.921368 

 

976.8095 

 

TG(20:3(5Z,8Z,11Z)/15:0/20:3(5Z,8Z,11Z)) 

 

C58H100O6 
 

23 

 

0.9064 

 

71.9484 

 

Superoxide 

 

O2 
 

24 

 

0.861032 

 

714.6696 

 

Cer(d18:1/23:0) 

 

C41H81NO3 
 

25 0.854799 69.9193 Propionic acid C3H2N2 



  

  82 J. Indonesian Trop. Anim. Agric. 48(2):76-88, June 2023 

Table 3. Identified putative metabolites in the testes (continued) 

 

No. VIP m/z Putative Metabolites Formula 

26 

 

0.854243 

 

1208.6346 

 

M(IP)2C(d18:0/16:0 

Preprosomatostatin 

C52H101NO24P2 
C52H83N17O15 
 

27 

 

0.836234 

 

136.9952 

 

Antharilic acid 

 

C7H7NO2 

 

28 

 

0.834576 

 

89.9993 

 

DL Lactic acid 

Lactic acid 

 

C3H6O3 

C3H6O3 

 

29 

 

0.826173 

 

705.8320 

 

Guanosine 3'-diphosphate 5'-triphosphate 

 

C10H18N5O20P5 

 

30 

 

0.820011 

 

1057.9136 

 

1-Heneicosanoyl-2-docosanoyl-3-sn glycerol 

 

C68H122O6 

 

31 

 

0.808577 

 

188.1263 

 

Antipyrine 

Gly-Leu 

 

C11H12N2O 

C8H16N2O3 

 

32 

 

0.796197 

 

137.0017 

 

Anthranilic acid 

Trigonelline 

 

C7H7NO2 

C7H7NO2 

 

33 

 

0.786638 

 

846.6861 

 

PC(18:0/22:0) 

PC(20:0/20:0) 

PC(16:0/24:0) 

PE(17:0/26:0)[U] 

PE(24:0/19:0)[U] 

PE(22:0/21:0)[U] 

 

C48H96NO8P 

C48H96NO8P 

C48H96NO8P 

C48H96NO8P 

C48H96NO8P 

C48H96NO8P 

 

34 

 

0.780037 

 

940.5459 

 

Dehydrosoyasaponin I 

 

C48H76O18 
 

35 

 

0.776376 

 

769.8285 

 

PE 38:3 

PC 35:3 

Phosphatidylcholine 17:1-18:2 

Phosphatidylethanolamine 18:0-20:3 

 

C43H80NO8P 

C43H80NO8P 

C43H80NO8P 

C43H80NO8P 

 

36 

 

0.768842 

 

1041.8005 

 

PS(24:0/24:1(15Z)) 

 

C54H104NO10P 

 

37 

 

0.735888 

 

54.4800 

 

Peroxynitrite 

 

HNO3 
 

38 

 

0.712305 

 

58.8516 

 

N4-Acetylaminobutanal 

 

C6H11NO2 
 

39 

 

0.684643 

 

120.0019 

 

(-)-1-(Methylthio)propyl 1-propenyl disulphide 

 

C6H12S2 

 

40 

 

0.647569 

 

132.0214 

 

L-Ornithine 

 

C5H12N2O2 

 

41 

 

0.63501 

 

51.1855 

 

Ethylbenzene 

 

C8H10 
 

42 

 

0.591421 

 

1116.0686 

 

TG(20:0/24:1(15Z)/22:1(13Z)) 

 

C69H130O6 
 

43 

 

0.562731 

 

59.8535 

 

Acetate 

 

C2H2O2 
 

44 0.527496 59.8654 Acetate C2H2O2 

 



 

 

                                                               

Rumen protected fat and reproductive genes in Malin rams (A. Ismail et al.) 83 

be attributed to have different metabolites from 

groups B, C, and D. 

 The variable importance for the projection 

(VIP) summarizes the importance of the varia-

bles 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 ar-

ger 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 varia-

bles 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. 

 The OPLS-DA plots of the metabolites in 

the testes differed between the control group (A) 

and the treatment group fed with RPF. This find-

ing indicates the presence of different metabo-

lites. There were 44 differentiated metabolites 

identified in testes using the VIP analysis 

(VIP>0.5). The important putative metabolites in 

this study are; 10 – hydroxy - (2E,8E) – decadi-

ene – 4 - ynoic acid (organic compounds known 

as medium- chain fatty acids), albendazole S-

oxide, Nicotinuric acid, Methylcyclopentane 

(saturated monocyclic hydrocarbons), CL (20: 1

(11Z) / 18: 2(9Z,12Z)/18: 1(11Z)/18:1 (11Z) 

glycerophospholipids, 18(R) hydroxy – 20 – oxo 

– 20 – CoA - LTE4 (metabolite through lipid oxi-

dation of Leukotriene E4 (LTE4)), CL(i-13:0/a-

21:0/i-24:0/a- 25:0)[rac]('double' phospholipids), 

Methylpentanoic acid (saturated fatty acids with 

an acyl chain that has a methyl branch). 

 In the control group (A), 10 – hydroxy - 

(2E,8E) – decadiene – 4 - ynoic acid was mainly                                   

identified. We found the metabolite in the group 

fed with Prilled fat (B) was different from the 

group (A), (C) and (D). The metabolite in group 

B was, 1 - Heneicosanoyl – 2– docosanoyl – 3 - 

(7Z, 10Z, 13Z, 16Z, 19Z, docosapentanoyl) – sn 

– glyceryl is categorized as Glycerolipids [GL], 

main class: Triradylglycerols [GL03] and sub-

class: Triacylglycerols [GL0301] (TAGs). The 

metabolites went through further changes how-

ever, we could not identify the specific metabo-

lites in group calcium salt and canola oil which 

 
EXACT MASS 

 
Figure 2. Viable importance for the projection (VIP) plot showed the summarized of the important metabolites 

(sorted from most important to lower) in the samples. 

VIP 
 



  

  84 J. Indonesian Trop. Anim. Agric. 48(2):76-88, June 2023 

are Adenosine 5' pentaphosphate and PE 

(24:0/24:1(15Z)). Adenosine is a product of 

complete dephosphorylation of adenine nucleo-

tides which are presence in various compart-

ments of the cell. PE (24:0/24:1(15Z))  is a phos-

phatidylethanolamine (PE or GPEtn). Fatty acids 

containing 16, 18 and 20 carbons are the most 

common, however PE (24:0/24:1(15Z)), in par-

ticular, 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 posi-

tions of glycerol within phospholipids are con-

tinually in flux, owing to phospholipid degrada-

tion and the continuous phospholipid remodeling 

that occurs while these molecules are in mem-

branes. PEs are neutral zwitterions at physiologi-

cal 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’ diphospha-

tase has been found in lactating bovine mamma-

ry glands. The enzyme is associated with mem-

branes of mitochondrial, microsomal, and Golgi 

Table 4. Identified putative metabolites in treatment groups. 

Groups* m/z Putative metabolites 

A 102.0377 10-hydroxy-(2E,8E)-decadien-4-ynoic acid 

B 1057.9136 1-Heneicosanoyl-2-docosanoyl-3-sn  glycerol 

C and D 705.8320 Adenosine 5' pentaphosphate 

  PE (24:0/24:1(15Z) 

*A- Control; B-Prilled fat; C- Calcium salt; D-Canola oil 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3.  Loading’s scatter (LS) plot illustrated the metabolites that consist in different treatment groups 



 

 

                                                               

Rumen protected fat and reproductive genes in Malin rams (A. Ismail et al.) 85 

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’ pen-

taphosphate represents an extremely useful tool 

in experiments with fragmented sarcoplasmic 

reticulum, such as studies of H
+ 

movement ac-

companying Ca
+
 movement, ATP-ADP ex-

change reaction, and calorimetry of the Ca
+
 up-

take process in bullfrog skeletal muscle. 

Cusostomo et al., (2020) reported that the in-

crease 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 comprehen-

sive classification system for lipids is comprised 

of eight lipid categories: Fatty acyls (FA), Glyc-

erolipids (GL), Sphingolipids (SP), Glycer-

ophospholipids (GP), Saccharolipids (SL), 

Prenol lipids (PR), Polyketides (PK), and Sterol 

lipids (ST). Each lipid category has its sub-

classification  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 triglycer-

ides. TAGs represent the most abundant lipid 

class in oils and fats of animal origin and com-

prise the bulk of storage fat in mammalian tis-

sue. These molecules exist as enantiomers since 

a centre of asymmetry is created upon enzymat-

ic biosynthesis at carbon 2 of the glycerol back-

bone (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 Jafaro-

ghli et al. (2014) who mention that lipids are 

abundant in testicles, playing a crucial role in 

membrane structure and function, energy stor-

age 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 cano-

la oil. CatSper4 was expressed up to four- fold 

in calcium salt meanwhile, in canola oil two-fold 

compared to control. The AdipoR2 gene was on-

ly 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 influ-

enced by testis development and spermatogene-

sis. 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 progres-

sive motility (Ahmad et al., 2018). SERPINA10 

gene can improve fertilization and sperm devel-

opment (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 spermatogen-

ic process  in the testes (Ma et al., 2013). The pre-

sent findings are congruent with the report made 

by Pavkovych et al., (2015), where supplementa-

tion of protected fats and polyenoic fatty acids 

of vegetable origin in a diet of cattle stimulates 

metabolism in the animals, increases their produc-

tivity, 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 ani-

mals. Present finding suggested that supplemen-



  

  86 J. Indonesian Trop. Anim. Agric. 48(2):76-88, June 2023 

tation of RPF in the form of calcium salt may 

improve the fertility of Malin ram through sperm 

total number, concentration, and progressive mo-

tility. 

 

CONCLUSION 

 

It could be concluded that feeding lipid as RPF 

with calcium salt had converted PUFAs to Aden-

osine, 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 indi-

cated the efficiency of the RPF metabolism to pro-

duce PUFAs, TAG, PE, and Adenosine during 

the spermatogenesis in the testes. The present 

study also revealed 44 important putative metab-

olites via preliminary screening of LC/MS. How-

ever, 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 me-

tabolites 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 im-

pression that supplementation of RPF with calci-

um salt will improve the reproductive efficiency 

in male ruminants by improving the motility of 

the                             sperm as well as the meiosis and spermatogen-

ic process in testes. 

 

ACKNOWLEDGEMENT 

 

 A.I. was a recipient of a scholarship from 

Federal Training Award (HLP), The Government 

of Malaysia. 

 

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