Alfa Enolase (ENO1) Sebagai Protein Antigenik Pada Spermatozoa Sapi Bali

Abstract

TEGUH SUMARSONO. Alpha Enolase (ENO1) as an Antigenic Protein in Bali Cattle Spermatozoa. Supervised by BAMBANG PURWANTARA, IMAN SUPRIATNA, MOHAMAD AGUS SETIADI and MUHAMMAD AGIL

Bali cattle are known for their high fertility. The reproductive success of cattle including Bali cattle, both of natural mating and artificial insemination depends on the ability of spermatozoa to fertilize oocytes. Among the factors that determine the spermatozoa ability is the presence of proteins associated with both the fertilization process and spermatozoa quality. Proteins present in spermatozoa, particularly those located on the surface of the plasma membrane, can bound by anti-spermatozoa antibodies, causing spermatozoa to lose their ability. One protein that has been detected as an antigenic protein in bovine spermatozoa is alpha enolase (ENO1), a glycolytic enzyme. The purpose of this study was to detect and categorize spermatozoa plasma membrane ENO1 and its presence to the quality of Bali cattle spermatozo. This study also aims to test the reactivity of ENO1 with IgG. In addition, this study was conducted to evaluate the quality of spermatozoa after elimination of ENO1 with anti- ENO1. This research was conducted in August – September 2020 and February – March 2021 at the Singosari Artificial Insemination Center and the Reproductive Rehabilitation Unit of Faculty of Veterinary Medicine, IPB University. The sample of this study consisted of 55 ejaculates/semen of Bali cattle obtained from 5 bulls. Other materials used were bovine IgG (MyBioSource, Sandiego, USA), anti-ENO1 (MyBioSource, Sandiego, USA), MAR-test kit (FertiPro, Belgium) and bovine ENO1 kit (MyBioSource, Sandiego, USA). The variables measured included total motility, progressive motility, viability, plasma membrane integrity (PMI), MAR value and alpha enolase quantity (ENO1). Measurement of motility and kinematics of mitility was performed using Computer-assisted Sperm Analysis (CASA; IVOS II, The Hamilton Thorne, USA). Viability of spermatozoa was measured using the eosin- nigrosine differential staining method. Plasma membrane integrity was measured using the Hypoosmotic Swelling test (HOS-test) method, the MAR value was determined using the Mix Antiglobulin Reaction test (MAR-test) method. ENO1 levels were determined the Enzyme Linked Immunosorbent Assay (ELISA) method. The experimental design used was a randomized block design with 2 IgG treatments (0 and 0.5 mg/ml) with 16 ejaculates groups, and 3 anti-ENO1 treatments (0, 50 and 100 ng/ml) with 9 ejaculates groups. Data analysis used analysis of variance (ANOVA) and if the treatment had a significant effect, it was followed up with Duncan's multiple range test. The relationship between the quantity of ENO1 and the spermatozoa quality parameters of was analyzed using Student's t-test. The results showed that ENO1 protein was found in the plasma membrane of spermatozoa obtained from normal ejaculate of Bali cattle at the level of 1.27 ng/106 spermatozoa. Motility rate did not affect the amount plasma membrane ENO1 (P<0.05). There was no correlation between total motility and the quantity ENO1 (P> 0.05; r = 0.21; t = 1.14), between progressive motility and the quantity of ENO1 (P> 0.05; r = 0 ,19; t = 1.01). Spermatozoa viability was also unrelated to the quantity of ENO1 (P> 0.05; r = 0.18; t count = 0.95). Analysis of the correlation between PMI

and the amount of ENO1 also yielded the results (P < 0.5; r = 0.18; t = 0.99. The results of the analysis of variance showed that plasma membrane ENO1 of Bali cattle spermatozoa can bind with IgG and Anti ENO1 causing a decrease in motility, viability, PMI, and plasma membrane ENO1 quantity and increasing the MAR value. The results of the study revealed that: 1) the presence of ENO1 wasa detected in the spermatozoa plasma membrane of Bali cattle; 2) in normal bull ejaculate/semen, plasma membrane ENO1 is not an antifertility substance and does not affect the spermatozoa quality of Bali cattle; 3) ENO1 in plasma membrane of spermatozoa is an antigenic protein; 4) Binding of ENO1 to the plasma membrane of spermatozoa may reduce spermatozoa quality in Bali cattle.

Key words : anti-ENO1, IgG, MAR Value, spermatozoa quality

Bali cattle are known for their high fertility. The reproductive success of cattle including Bali cattle, both of natural mating and artificial insemination depends on the ability of spermatozoa to fertilize oocytes. Among the factors that determine the spermatozoa ability is the presence of proteins associated with both the fertilization process and spermatozoa quality. Proteins present in spermatozoa, particularly those located on the surface of the plasma membrane, can bound by anti-spermatozoa antibodies, causing spermatozoa to lose their ability. One protein that has been detected as an antigenic protein in bovine spermatozoa is alpha enolase (ENO1), a glycolytic enzyme. The purpose of this study was to detect and categorize spermatozoa plasma membrane ENO1 and its presence to the quality of Bali cattle spermatozo. This study also aims to test the reactivity of ENO1 with IgG. In addition, this study was conducted to evaluate the quality of spermatozoa after elimination of ENO1 with anti- ENO1. This research was conducted in August – September 2020 and February – March 2021 at the Singosari Artificial Insemination Center and the Reproductive Rehabilitation Unit of Faculty of Veterinary Medicine, IPB University. The sample of this study consisted of 55 ejaculates/semen of Bali cattle obtained from 5 bulls. Other materials used were bovine IgG (MyBioSource, Sandiego, USA), anti-ENO1 (MyBioSource, Sandiego, USA), MAR-test kit (FertiPro, Belgium) and bovine ENO1 kit (MyBioSource, Sandiego, USA). The variables measured included total motility, progressive motility, viability, plasma membrane integrity (PMI), MAR value and alpha enolase quantity (ENO1). Measurement of motility and kinematics of mitility was performed using Computer-assisted Sperm Analysis (CASA; IVOS II, The Hamilton Thorne, USA). Viability of spermatozoa was measured using the eosin- nigrosine differential staining method. Plasma membrane integrity was measured using the Hypoosmotic Swelling test (HOS-test) method, the MAR value was determined using the Mix Antiglobulin Reaction test (MAR-test) method. ENO1 levels were determined the Enzyme Linked Immunosorbent Assay (ELISA) method. The experimental design used was a randomized block design with 2 IgG treatments (0 and 0.5 mg/ml) with 16 ejaculates groups, and 3 anti-ENO1 treatments (0, 50 and 100 ng/ml) with 9 ejaculates groups. Data analysis used analysis of variance (ANOVA) and if the treatment had a significant effect, it was followed up with Duncan's multiple range test. The relationship between the quantity of ENO1 and the spermatozoa quality parameters of was analyzed using Student's t-test. The results showed that ENO1 protein was found in the plasma membrane of spermatozoa obtained from normal ejaculate of Bali cattle at the level of 1.27 ng/106 spermatozoa. Motility rate did not affect the amount plasma membrane ENO1 (P<0.05). There was no correlation between total motility and the quantity ENO1 (P> 0.05; r = 0.21; t = 1.14), between progressive motility and the quantity of ENO1 (P> 0.05; r = 0 ,19; t = 1.01). Spermatozoa viability was also unrelated to the quantity of ENO1 (P> 0.05; r = 0.18; t count = 0.95). Analysis of the correlation between PMI and the amount of ENO1 also yielded the results (P < 0.5; r = 0.18; t = 0.99. The results of the analysis of variance showed that plasma membrane ENO1 of Bali cattle spermatozoa can bind with IgG and Anti ENO1 causing a decrease in motility, viability, PMI, and plasma membrane ENO1 quantity and increasing the MAR value. The results of the study revealed that: 1) the presence of ENO1 wasa detected in the spermatozoa plasma membrane of Bali cattle; 2) in normal bull ejaculate/semen, plasma membrane ENO1 is not an antifertility substance and does not affect the spermatozoa quality of Bali cattle; 3) ENO1 in plasma membrane of spermatozoa is an antigenic protein; 4) Binding of ENO1 to the plasma membrane of spermatozoa may reduce spermatozoa quality in Bali cattle. Key words : anti-ENO1, IgG, MAR Value, spermatozoa quality