SYNERGISTIC EFFECT OF SPERM FILTRATION TECHNIQUES AND ANTIOXIDANTS ADDED TO EXTENDER OF IRAQI BUFFALO SEMEN

This study investigated the synergistic effect of glass wool and sephadex sperm filtration techniques on improving the low semen quality of buffalo bulls with or without antioxidants. Semen was collected for 14 weeks, evaluated weekly, and divided into 12 groups. Good and low semen have extended using a Tris extender. Good semen quality is divided into three groups [S1: Tris extender; S2: Tris + vitamins E (2 mM) and C (5 mM); S3: Tris + glutamine (20 mM) and arginine (1 mM)]. Low semen quality was divided into three main groups and subdivided into three sub-groups (S4; Tris extender; S5: Tris +vitamins E (2 mM) and C (5 mM); S6: Tris + glutamine (20 mM) and arginine (1 mM)). In the 2 nd and 3 rd main groups, glass wool and sephadex techniques are used with or without adding vitamins (E+C) and amino acids (Glutamine and arginine) and subdivided into three sub-groups with each technique, referred to as S7–S9 for glass wool and S10–S12 for sephadex techniques. Improving (P<0.01) motility, normal morphology, acrosome, and plasma membrane integrity and reducing total sperm abnormalities of low semen quality were noticed using sephadex with or without adding vitamins or amino acids post-cryopreservation. In conclusion, the sephadex technique removed immotile sperm, dead, abnormal, and good sperm harvested by this technique, which in turn may reflect positively on improving the pregnancy rate.


INTRODUCTION
Semen is affected by many factors, including genetics, nutrition, diseases, age, management, vaccination, stress, transportation, and others (9,13,16,21,27,36).There is a strong relationship between fertility and semen characteristics fresh of buffalo bulls (7,8,37,38) and bovine bulls (6,16,17).Buffalo is a low-grade reproductive efficiency animal (4).The females' artificial insemination using lowfertility bull semen leads to a significant loss for the dairy buffalo industry.Therefore, it is necessary to find reliable tests or tools to improve the semen quality of buffalo bulls or to determine the latent fertility of buffalo bulls.The bulls used in artificial centers do not always produce good-quality semen (35).The artificial insemination centers are a huge burden or an economic loss that is not insignificant, estimated at millions of $ due to the use of sperm from low-fertility bulls.These losses are estimated at 3-4 $/day/bull in Iraq (24) and had negatively reflected in the low financial return of breeders and artificial insemination centers.Thus, improving semen quality is necessary to get the most benefit from genetically-superior bulls or/and have poor quality semen.The sperm filtration techniques significantly improved semen quality in vitro fertilization.Filtration techniques have become very important in the processes of in vitro fertilization and artificial insemination.In semen filtration techniques, motile sperms filtered from debris, non-motile, and abnormal.Sperm filtration techniques include dilution and washing, selective filters such as glass wool, Sephadex (5,10,24,26), density gradient centrifugation such as Bovipure (43) Sill Select Plus (11,32,33), and self-migration techniques such as the swimdown or swim-up technique (25,26,27,33,34).Some techniques of semen filtration from fresh semen or post-thawing semen gave good results for bull semen characteristics (24, 25,26).The plasma membrane of buffalo sperm has a higher percentage of polyunsaturated fatty acids than that of bovine bulls (42), which makes it more susceptible to oxidative stress during cryopreservation.Thus, there is a decrease in motility and the integrity of the plasma membrane, acrosome, and chromatin in buffalo sperm.This weakness is mainly due to the low concentrations of antioxidants in buffalo semen.Antioxidants have been reduced during the cryopreservation and thawing processes.The continuous production of free radicals in the seminal plasma from abnormal, immature, dead sperm, and cryopreservation processes had accompanied by a decrease in antioxidants concentrations in the seminal plasma, which has a significant effect on bull semen quality (3,8,19,20,22,39), which leads to oxidative stress on the sperm.The addition of some antioxidants to semen bulls gave good results in improving the semen quality postcryopreservation (7,8,15,19,22).Vitamins E and C are antioxidants and act as inhibitors of the oxidation of unsaturated fatty acids in the sperm plasma membrane through their ability to inhibit free radicals very efficiently and can act synergistically (17).Numerous studies have indicated an improvement in the characteristics of bull semen after cryopreservation when vitamin E or C had added to the semen diluents (18, 29).The glutamine plays an antioxidant and protects sperm from freezing damage in bulls (2).It found that glutamine, egg yolk, and glycerol act synergistically in bull semen diluents to prevent freezing damage.Abdel-Razek and El-Shamaa (1) showed that the addition of glutamine to semen diluents of buffalo bulls improved individual motility, viability, integrity of the sperm plasma membrane, and fertility rate.It also reduced the formation of reactive oxygen species inside the sperm compared with the sperm of the control group.Arginine is an amino acid that plays a crucial role in sperm motility.A decrease in arginine concentration causes disturbance of sperm metabolism, lack of sperm motility, spermatogenesis, and oxidation of sperm membranes (12), as it is associated with free radicals.Badr (12) found that the addition of 0.5 mmol of arginine to the semen diluent of Egyptian buffalo bulls improved the individual motility and viability of sperm and the pregnancy rate with a decrease in the concentration of malondialdehyde compared with the control group.The amino acids arginine and glutamine act synergistically as antioxidants in cells.The semen filtration techniques for low-grade-quality semen from buffalo bulls have not been addressed previously in Iraq.This study has been conducted to investigate the effect of using glass wool and sephadex techniques to filtrate low-quality semen from Iraqi buffalo bulls.The use of antioxidants (vitamin C with E or glutamine with arginine) may give good results in improving or activating the filtered or unfiltered semen through one of the filtration technologies for buffalo bulls by taking advantage of both and the possibility of preserving it by freezing and using it for commercial purposes (increasing the number of straw produced by the artificial insemination center).

MATERIALS AND METHODS Experimental design
The study carried out at the Artificial Insemination Department/ Directorate of Animal Resource, Ministry of Agriculture.Ten Iraqi buffalo bulls (500-750 kg weighting and 6-8 years old) were trained for semen collection using an artificial vagina.The experimental buffalo bulls have good health.Bulls with low-quality fresh semen (n=6; individual motility ≤40%) and good-quality (n=3; individual motility ≥50%) were collected via artificial vagina at one ejaculate/buffalo bull/week for 14 weeks.Both low and good-quality semen divided into 12 groups.Good and low semen was extended using a Tris extender.Good semen quality was divided into three groups [S1; Tris extender, S2; Tris+ vitamins E (2 mM) and C (5 mM), S3; Tris +glutamine (20 mM) and arginine (1 mM)].Low semen quality was divided into three main groups and subdivided into three sub-groups (S4; Tris extender, S5; Tris +vitamins E (2 Mm) and C (5mM), S6; Tris + glutamine (20 mM) and arginine (1 mM)].In the 2 nd and 3 rd main groups, glass wool and sephadex techniques used with or without adding vitamins (E+C) and amino acids (Glutamine and arginine) and subdivided into three sub-groups with each technique and referred to as S7-S9 for glass wool and S10-S12 for sephadex technique.All buffalo bulls were allocated on a standardized diet, as a concentrate ration (protein18% and 2146 kcal) was provided daily at a rate of 4-6 kg/bull.Roughage consisted of alfalfa hay (7-9 kg/bull/day) besides green forage (50-60 kg/bull/day).Fresh water and salt blocks were available ad libitum to the bulls.

Semen characteristics
The semen characteristics had evaluated (45) post-cooling and cryopreservation (sperm's cell individual motility, live sperm, normal morphology, sperm's acrosome, and plasma membrane integrity, total antioxidants and malondialdehyde concentrations, DNA damage, and freezability).

Statistical analyses
The statistical computation had performed using the SAS program based on a study on completely randomized design (CRD) to study the effect of different factors in the study characteristics.Means with significant differences had compared using Duncan multiple range test.The statistical models for comparison among groups Yij = µ + Ti + eij Yij = dependent variable pertaining to the j observation of the i treatment µ = overall mean Ti = effect of i treatment (i-positive, negative controls, glass wool, sephadex techniques) eij = error term.

Post-cryopreservation
The results of sperm cell individual motility exhibited significant differences (P≤0.01)among groups post-cryopreservation period of buffalo bulls' semen (Table 2).Greater individual motility was recorded in the S1-S3 groups (36.25-38.33%)while lesser in the S4 group (15%).The S1-S3 groups revealed higher (P≤0.01)live sperm than the rest groups.The S10-S12 groups recorded higher (P ≤ 0.01) normal morphology (%) than the S1-S8 groups.The results demonstrated significant differences among groups in acrosome and plasma membrane integrity (Table 2).A high (P ≤ 0.01) amount of total antioxidant activity has found in the S3 group as compared with the S1-S2, S5-S9, and S11 groups (Figure 1A).Less (P ≤ 0.01) DNA damage percentages were found in the glass wool (S7) and sephadex (S10) filtration groups than in the S12 group (Figure 1B).The S6-S12 groups revealed lower (P ≤ 0.01) malondialdehyde concentrations than the S1-S5 groups (Figure 1C).The S9 group exhibited higher freezability, while the S4 group was less so (Figure 1D).Also, the S9 group exhibited higher (P ≤ 0.01) freezability than the S1-S8 groups (Figure 1D).The superiority of S1-S3 treatments in individual motility, live sperm, acrosome, and plasma membrane integrity (Tables 1 and 2) over most experimental treatments is because the quality of the semen used for cryopreservation was good.There are several antioxidants in semen, some of which are found in the nucleus (vitamin E), lysosomes (vitamin C and E, and carotenoids), mitochondria (such as vitamin E, C, manganese, catalase, and glutathione peroxidase), cytoplasm (vitamin C, E, and glutathione peroxidase), and cell membranes.However, these antioxidants are present in small amounts (40), and their sufficient presence in mammalian sperm prevents and reduces oxidative damage to the lipid membranes of sperm during cryopreservation and thawing processes.Non-significant differences in most semen characteristics post cooling and cryopreservation (Tables 1 and 2) were found in the current study among the S1-S3 groups.Contrary to our results of the cryopreservation of bull semen, Eidan and Al-Zaidi (18) reported that adding a combination of vitamin C and E to the semen extender improved acrosome integrity and reduced midpiece abnormalities as compared with control group.We suppose the lack of significant differences in most semen characteristics in the current study among S1-S3 groups may be a return of several reasons: First, the amount of vitamin E with C (S2) was insufficient, or the semen of buffalo bulls didn't suffer from a deficiency of glutamine with arginine acids (S3) as mentioned in Figure 1A.The second reason is that buffalo semen may need treatment or the addition of other antioxidants like enzymatic, non-enzymatic, and natural antioxidants (7,8,15,16,17,19,24,25,28,34,39).A third reason may be a need for genetic improvement by selecting genes related to semen characteristics (9,21,44,46,47).Sperm individual motility percentage had frequently been determined in artificial insemination laboratories, an essential requirement in oocyte fertilization (14).The cervical mucus selectively allows only progressively motile sperm of normal morphology to penetrate and migrate through the cervix (45) .The sephadex filtration with or without antioxidants (S10-S12) improved most of the semen traits in this study than the negative control group (S4; Table 1, 2 and Figure1).Semen filtration technology through Sephadex columns (G10-G200) which is a dextran gel, was used in bulls (31) and buffaloes (25).This technology relies on the passage of normal morphology of sperm and motile through the pores between the sephadex particles and their collection at the bottom of the collecting tube with the accumulation of dead and non-motile sperm on the surface of the sephadex gel (6).Any improvement in the percentages of sperm motility has regarded as a normal response for sperm activity post the removal of seminal plasma since it contains dead and abnormal sperms, leukocytes, epithelial cells, debris, and microbial contamination that produce many oxygen radicals that negatively influence the sperm functions.Mammalian fertilization and subsequent embryonic development depend on the integrity of the sperm's DNA (41).Understanding the complex packaging of mammalian sperm chromatin and the assessment of DNA integrity could potentially provide a benchmark in clinical infertility (30,41).The DNA damage percentage in this study was still within the permissible (Figure 1B) limit value (7-20%) for a bull's fertility (23,40), indicating good bull fertility.The S7 (Glass wool group) and S10 (Sephadex group) recorded the lowest DNA damage sperm percentages than other groups (Figure 1C), which may be because glass wool filtration has mainly based on the fact that due to the sticky nature of dead, non-motile, and abnormal sperm, they easy get attached to fiber (26,45).Also, during the sephadex filtration process, dead and non-motile sperms accumulate on the surface of the sephadex gel (26,45).A higher (P ≤ 0.01) freezability recorded in the S9 group than the S1-S8 groups (Figure 1D) may be due to the fact that arginine and glutamine act synergistically as antioxidants and reduce the production of free radicals, which form due to oxidative stress during the cryopreservation process.Although most of the sperm filtration treatments recorded significantly or mathematically higher characteristics of the semen than a negative control, they were not within the level required for artificial insemination, perhaps due to the long period of sperm filtration within each technology, which causes the sperm to suffer from high acidity in the filtering tubes as a result of the metabolic processes that it performs before cooling and cryopreservation, which affects the motility of the sperm and membrane integrity.Therefore, we need to reduce the filtration time of the sperm or add substances that modify