Protein Profiles in Seminal Plasma of Iraqi Buffalo Bulls (Bubalus bubalis) Associated with Fresh and Cryopreserved Semen Quality

This study was undertaken to identify some protein profiles in the seminal plasma of Iraqi buffalo bulls and their correlation to the quality of fresh and cryopreserved semen. Semen was collected from eight buffalo bulls and divided according to the percentage of sperm individual motility into two groups. The first group (n=3) included good semen quality, while the second group (n=5) included poor semen quality. Seminal plasma was extracted from each semen sample by centrifugation and seminal proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nine types of protein bands were observed in the seminal plasma of the buffalo bulls belonging to good semen quality, ranging from 10-68 kDa. The most abundant proteins in the overall presence of proteins in seminal plasma were 10 (100%), 14 (100%), 20 (100%), 26 (100%), 34 (100%), and 43 (37.5%) kDa. On the other hand, 14 types of protein bands resolved for the poor-quality semen, ranging from 10-155 kDa. The most abundant proteins in the overall presence of proteins in seminal plasma were 10 (100%), 14 (100%), 20 (100%), 26 (100%), 34 (87.5%), 130 (50%) and 155 (50%) kDa. A significant negative correlation was noticed among proteins with molecular weights of 28. 88 ± 3.26, 22.50 ± 2.85, and 12.00 ± 1.15 kDa, and the percentage of sperm individual motility and plasma membrane integrity. Moreover, there was a positive significant correlation between proteins whose molecular weight 22.50 ± 2.85 kDa and the percentages of total abnormalities and normal sperms. In conclusion, the identification of seminal proteins may be a useful tool in the future evaluation of spermatozoa and the prediction of fertility in Iraqi buffalo bulls.


Introduction
The prediction of the fertility of bulls is one of the factors of great importance in reaching the optimum efficiency in the world dairy industry systems.The assessment of the fertility of bulls is one of the main challenges facing artificial insemination centers and owners of cows and buffaloes, and the semen measurements adopted in these centers are not reliable globally to determine the fertility of bulls at present [1,2].The field fertility of bulls is affected depending on the pregnancy rate of cows and buffaloes by several factors, including the health status of the bulls, genetic characteristics, the capacity of the reproductive organs, herd management, semen quality, and the steps used for sperm cryopreservation [1,3,4,5,6].On the other hand, biomarkers such as various proteins play an essential role in the biological systems of bulls, but detailed information about their presence in seminal plasma, 2 their importance in sperm metabolism, and their relationship to the fertility of buffalo bulls have not been widely studied [7][8][9][10][11].
The seminal proteins play principal roles in sperm protection, capacitation, acrosome reaction, spermegg binding, fertilization, and early embryonic development [12].Therefore, the identification of these indicators in the seminal plasma of good and poor-quality semen will give us additional information about the possibility of predicting the fertility of buffalo bulls through their role in sperm metabolism during their formation stages.Dixit et al [13] found 24 types of proteins in the seminal plasma of Bhadawari buffalo bulls in India, with weights ranging from 6 to 200 kDa.Similarly, Sharma et al [14] found a correlation between seminal plasma proteins and the quality of good quality semen of Bhadawari buffalo bulls in India however, they did not address the relationship of these proteins to poor quality semen or comparison with good quality.On the other hand, Fu et al [15] analyzed the proteins of buffalo sperm and seminal plasma.About 2147 and 864 proteins were identified in mature sperm and seminal plasma.The ODF2, AKAP4, and TUBB were the most abundant proteins in spermatozoa, while the three most abundant proteins were in plasma ALB, CLU, and AZGP1.Molecular genetic analysis indicated that most sperm proteins of buffalo bulls were involved in transport, phosphorylation, and pathways related to energy metabolism, protein processing, and RNA transfer significantly in sperm, while those belonging to the seminal plasma (Protease and lysosomal activities) also play a crucial role in sperm protection [15].Moreover, proteins in the seminal plasma of bulls are potential biomarkers of sperm tolerance to cryopreservation [16].
The absence of previous studies concerned with detecting the profile of seminal proteins and their relationship to the poor and good quality of fresh and cryopreserved semen for Iraqi buffalo bulls prompted us to undertake the current work.

Experimental animals and design
This study was carried out at the Department of Artificial Insemination belonging to the Directorate of Animal Resources, Iraqi Ministry of Agriculture, Baghdad, from December 2021 to October 2022 using eight Iraqi buffalo bulls (3.5-5 years old and 500-750 kg live body weight).All bulls were in good health and under continuous veterinary supervision.All animals fed 5 kg of concentrate diet/animal/day consisted of 35% barley, 33% wheat bran, 10% yellow corn, 20% soybean meal, 0.5% limestone, and 1% vitamins and minerals.The crude protein percentage and total energy amount of the diet were 18% and 2164 kilocalories/kg, respectively.Concomitantly, bulls provided 7-9 kg of hay and 40-50 kg of alfalfa per animal/day.Semen was collected from each bull via an artificial vagina and divided into two groups according to their semen quality; good (n=3; sperm individual motility 50 % and above) and poor (n=5; sperm individual motility less than 50 %).

Semen sampling and evaluation
Semen was collected as one ejaculate per week per bull for 8 weeks.Good and poor-quality semen was evaluated for fresh, three, and six-month post-cryopreservation (PC).Sperm cell individual motility [17,18], live sperm [19], abnormal sperms [20], sperm plasma membrane [21], and acrosome integrity [22] percentages were estimated for each group for the fresh, 96 hr, three, and six months' postcryopreservation (PC).The total number of perms per semen straw is calculated according to Eidan [19].

Identification of protein profiles in seminal plasma
One milliliter from each ejaculate was collected and centrifuged to separate the seminal plasma from the sperm at a rate of 3000 rpm for 15 minutes.The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed (13.5%) to characterize the seminal proteins based on their molecular weight [23].The molecular weight was estimated by comparison with standard molecular weight proteins ranging from 10-250 kDa (Merck, Mumbai, India).

Statistical Analysis
The statistical analysis system [SAS; 24] was used to analyze the data based on complete randomized design (CRD) to study the relationship between the types of proteins in the seminal plasma and semen quality.The statistical model of the study was as follows: Yij = µ + Fi + eij Yij = dependent variable pertaining to the j observation of the i group.µ = overall mean.Fi = effect of semen quality (Good or bad).eij = error term.Means with significant differences were compared using Duncan multiple range test [25].Exponential equations were applied according to the simple non-linear regression analysis by calculating and determining the coefficient of determination (R2).

Electrophoretic profile of proteins in seminal plasma
SDS-PAGE analysis identified nine types of protein bands in the seminal plasma of the buffalo bulls belonging to good semen quality (GS), ranging from 10-68 kDa.The most abundant proteins in the overall presence of proteins in seminal plasma were 10 (100%), 14 (100%), 20 (100%), 26 (100%), 34 (100%), and 43 (37.5%)kDa, while the lesser existed types of protein were 48, 55 and 68 kDa (12.5%;Table 1 and Figure 1).For the poor-quality semen (PS), 14 types of protein bands resolved, ranging from 10-155 kDa.The most abundant proteins in the overall presence of proteins in seminal plasma were 10 (100%), 14 (100%), 20 (100%), 26 (100%), 34 (87.5%),130 (50%) and 155 (50%) kDa, while the lesser existed types were 50 (37.5%),45 (25%), 30 (12.5%), 40 (12.5%),49 (12.5%) and 112 kDa (12.5%;Table 1 and Figure 1).Moreover, proteins of high molecular weights (112, 130, and 155 kDa) were revealed in the PS other than the GS group (Table 1 and Figure 1).It is noteworthy that protein bands of 10, 14, 20, 26, 34, and 43 kDa were more predominant in the GS group while, the 10, 20, 26, 34, 45, 50, 130, and 155 kDa were predominant in a PS group (Table 1and Figure 1).Protein bands of 10, 14, 20, and 34 kDa were larger and denser in the seminal plasma of the GS group, whereas it was smaller in that of the PS group (Figure 1).Excluding 133 and 155 kDa proteins which were higher (P≤0.05) in PS than the GS groups, the differences between the two groups in other protein types lacked significance (Table 2).This is the first study concerned with the relationship of protein in seminal plasma with the semen quality of Iraqi buffalo bulls.The bull's fertility is a multifactorial attribute depending on several factors including semen quality [26,27].These results agreed with those reported by Almadaly et al [28] who found ten protein bands in the seminal plasma of fertile and sub-fertile Egyptian buffalo bulls.Moreover, Harshan et al [29], Asadpour et al [30], and Arangasamy et al [31], who found 19 bands (3-250 kDa), 25 bands (14.4-80.5 kDa), and 18 bands (12-127 kDa) respectively in seminal plasma of different buffalo bull's breeds.On the other hand, Baruti et al [32] found seven bands of proteins in the seminal plasma of the swamp buffalo with different molecular weights (25,28,38,55,65,71,and 161 kDa).Protein bands of 25-104 kDa in the seminal plasma were evidence of high-fertile bulls.Moreover, the protein of 55 kDa was Osteopontin, and those of 29-30 kDa were heparin-binding proteins.Further, Ivanova et al [33] revealed that the protein bands of 26 and 55 kDa were predominant in the seminal plasma of high-fertile buffalo bulls, whereas those of 15-16 kDa were common in the seminal plasma of low-fertile buffalo bulls.Accordingly, the 26 and 55 kDa bands were detected in the seminal plasma of goodsemen quality bulls in the current study (Table 1 and Figure 1).It is worth mentioning that the seminal plasma of the buffalo bulls contains substances that have a crucial role in the biological characteristics of the sperms.These substances were correlated with the high freezability of the sperms.It was also found that freezing-tolerant ejaculates of the buffalo bulls have a protein band of 6.4-14 kDa, whereas these proteins didn't exist in the freezing non-tolerant ejaculates [33].Sperm characteristics of buffalo bulls with good and poor-semen quality.
1. Fresh semen Non-significant differences were observed between GS and PS groups in ejaculate volume (EV) and sperm concertation (SC; Table 2).The GS group exhibited higher (P≤0.0001)percentages of sperm mass activity (MA), individual motility (IM), live sperm (LS), plasma membrane integrity (PMI), acrosome integrity (AI), and normal morphology (NM) as compared with the PS group (Table 2).On the other hand, a profound decline was noticed in sperm head abnormalities (SHA; P≤0.0004), tail principal and terminal abnormalities (TPTA; P≤0.002), and total abnormalities (TA; P≤0.0001) for GS as compared with the PS groups (Table 2).Non-significant differences were shown between GS and PS groups in sperm tail midpiece abnormalities (TMA; Table 2).Concomitantly, the differences between GS and PS groups lacked significance in the total number of motile sperms (TMS), plasma membrane integrity (TPMI), acrosome integrity (TAI), fraction sperm fraction (TFSF) and normal sperm (TNS; Table 2).
Similar ejaculate volume (3.02 ± 0.14 ml) was found by Sharma et al [34] and lesser than those reported by Tapaloaga et al (35; 3.85 ± 0.18 ml) for buffalo bulls when compared with the current results.On the other hand, lesser sperm concentrations obtained currently (for GS and PS groups) than those reported by Sharma et al [34; 1079.23 ± 16.0 ×10 6 ] in buffalo bulls.Differences among studies may return to the variation in the management practices and feeding levels and protocols of the bulls [2,36,37].It is worth mentioning that reducing the feeding levels causes decreasing levels of plasma calcium.Calcium is a crucial factor for sperm motility and fertility of the bulls [18,38].Further, the reasons behind non-significant differences in EV, SC, TMS, TPMI, TAI, TFSF and TNS between GS and PS groups may be attributed to the similar ages of the experimental animals, and being of the same breed, which was subjected to the similar management and nutritional conditions [39].

Ninety-six hr. post-cryopreserved semen
Non-significant differences were observed between GS and PS groups in the percentage of sperm IM of buffalo bulls, 96 hr.post-cryopreservation (PC), whereas the GS group was superior (P≤0.005) in LS percentage (49.26± 3.17 %) than the PS group (39.4 ± 1.71%; Table 3).There were non-significant differences between the GS and PS groups in the percentage of SHA, TPTA, and TA.In contrast, the TMA percentage decreased (P≤ 0.001) in the GS group compared to the PS group (Table 3).Concomitantly, the GS group recorded higher (P≤0.002)percentages of the sperm PMI (48.6 ± 2.98%) and AI (51.4 ± 3.02%) compared to the PS group (37.44 ± 1.92 and 40.12 ± 1.93% respectively) at 96 hr PC (Table 3).The differences between the GS and PS groups in the NS percentage and the TMS lacked significance.Also, the GS group recorded a higher (P≤ 0.002) TPMI and TAI compared with the PS group (Table 3).

Three months' post-cryopreservation
A higher (P≤0.001)percentage of sperm IM (27.66 ± 3.30 vs. 17.6 ± 1.04%) and LS (45.33 ± 3.20 vs. 34.8± 1.54%) has been detected for GS than PS groups, three months PC (Table 4).Moreover, a lesser (P≤0.004)percentage of SHA has been noticed for GS (4.8 ± 0.43%) than PS (6.28 ± 0.27%) groups within similar preservation periods (Table 4).Concomitantly, the GS group exhibited lesser (P≤0.02)TPTA and TA than the PS group, whereas the differences observed between the two-mentioned groups in TMS, three months PC lacked significance (Table 4).Further, greater (P≤0.0001)percentages of SPMI, SAI, and NS (P≤0.003) were shown for GS than for PS groups, three months PC (Table 4).The GS group exhibited a higher (P≤0.0001)TMS, sperms with TPMI and TAI, NS, and TFSF than those of the PS group, three months PC (Table 4).Table 2. Comparison of fresh good and poor-semen characteristics of Iraqi buffalo bulls using microscopic examination method (Mean ± SE).
Means with different superscripts within each row indicate significant differences (P≤0.0001).NS: Non-significant.Means with different superscripts within each row indicate significant differences.NS: Non-significant.
Table 5.Comparison of good and poor-quality semen of Iraqi buffalo bulls six months postcryopreservation using microscopic examination methods (Mean ± SE).
Means with different superscripts within each row indicate significant differences.NS: Non-significant.

The correlation coefficient between protein types in seminal plasma and semen characteristics
A negative and significant (P≤0.01)correlation coefficient was observed between PF1 proteins (111.50 ± 18.0 kDa) and the percentage of LS (r=-0.85)and AI (r=-0.88).Moreover, the correlation was negative and significant (P≤0.05) between PF1 proteins and the percentage of NS (r=-0.74;Table 6).The correlation coefficient was positive and significant (P≤0.05) between PF1 proteins and the TA

Level of significance
Poor-quality semen (n=5) Good-quality semen (n=3) Semen characteristics P≤0.0003 percentage (r=0.74).Non-significant and weak correlation coefficients were shown between PF1 and the remaining semen characteristics of Iraqi buffalo bulls (Table 6).The correlation of PF2 proteins (87.63 ± 16.5 kDa) with the percentage of LS was negative and significant (P≤0.05;r=-0.79).Similarly, a negative and significant (P≤0.01)correlation was observed between the PF2 proteins and sperm AI percentage (r=-0.86).The correlation coefficient between PF2 protein and remaining sperm attributes lacked significance (Table 6).A negative and significant (P≤0.05)correlation coefficient was shown between PF3 proteins (43.63 ± 3.14 kDa) and LS percentage (r=-0.62).The correlation between these protein types and other semen characteristics lacked significance and strength (Table 6).Similarly, the correlation coefficient between PF4 proteins (36.13 ± 3.33 kDa) and the percentage of both LS (r=-0.79)and sperm AI (r=-0.76)was negative and significant (P≤0.05).However, these correlations between PF4 and the remaining sperm characteristics of Iraqi buffalo bulls were weak and non-significant (Table 6).
The negative and significant correlations between PF8 (14.66 ± 1.84 kDa) and sperm LS percentage revealed the negative role of spermadhesion phospholipase (A2) protein (14 kDa) in these attributes.However, this protein participates in the capacitation, acrosome reaction, and sperm-oocyte membrane fusion [40].Furthermore, the presence of protein in the seminal plasma of the bovine and bubaline seminal plasma is associated with fertility [41].Excluding data from one buffalo bull in the GS group, Osteopontin protein (OPN; 55 kDa) was absent in the seminal plasma of the other bulls either in GS or PS groups (Table 1).This means the absence of the beneficiary effect of OPN in sperm fertilizing capacity in Iraqi buffalo bulls.It is well-known that the OPN concentration in the bovine seminal plasma was related to in vivo fertility of Holstein bulls [41] and to the fertilizing capacity of cauda epididymal sperm treated with the secondary sex gland fluids in IVF experiment [42].OPN is secreted by the accessory sex glands, binds to the post-ejaculated sperm, and contact with the secretion of the oviduct during capacitation [43].On the other hand, the presence of the T-cadherin protein (130 kDa) in the seminal plasma of the PS group (Table 1) revealed the diverse impact of this protein on fertilizing ability of the Iraqi buffalo bulls.The T-cadherin was involved in improper biological processes like the negative guidance of the motor axons and hind limbs trajectory during embryonic development [44].

Conclusion
The identification of seminal proteins may be a useful tool in the future evaluation of spermatozoa and the prediction of fertility in Iraqi buffalo bulls.However, most protein types in seminal plasma have a negative relationship with the semen characteristics.

Semen characteristics
Proteins (χ 2 )Values in parentheses indicate the percentage of either bad and good-semen quality or total, − absence, + presence.NS=Non-significant.

Figure 1 .
Figure 1.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in seminal plasma proteins of good and bad-quality semen of Iraqi buffalo bulls.

Table 1 .
Electrophoretic profiles of seminal plasma proteins for good and poor-quality semen of Iraqi buffalo bulls.

Table 3 .
Comparison of good and poor-quality semen characteristics of Iraqi buffalo bulls 96 hr post-cryopreservation using microscopic examination methods (Mean ± SE).

Table 4 .
Comparison of good and poor-quality semen of Iraqi buffalo bulls three months' postcryopreservation using microscopic examination methods (Mean ± SE).

Table 6 .
The correlation coefficient between semen characteristics of Iraqi buffalo bulls with different types of proteins in seminal plasma (kDa).: P≤0.05.* *: P≤0.01.PF1: protein type at a molecular weight of 111.5 ± 18.0 kDa PF2: protein type at a molecular weight of 87.63 ± 16.5 kDa.PF3: protein type at a molecular weight of 43.63 ± 3.14 kDa.PF4: protein type at a molecular weight of 36.13 ± 3.33 kDa.PF5: protein type at a molecular weight of 28.88 ± 3.26 kDa.PF6: protein type at a molecular weight of 22.50 ± 2.85 kDa.PF7: protein type at a molecular weight of 18.57 ± 2.34 kDa.PF8: protein type at a molecular weight of 14.66 ± 1.84 kDa.PF9: protein type at a molecular weight of 12.00 ± 1.15 kDa.