View of Study Motility of Ongole Crossbred Semen with Soybean-Based Diluents Stored at Ambient Temperature (24-27 0C)

Study Motility of Ongole Crossbred Semen with Soybean-Based Diluents Stored at Ambient Temperature (24-27⁰C)

Dian Ratnawati^*1,2), Gatot Ciptadi¹⁾, Sri Rahayu³⁾ and Trinil Susilawati¹⁾

1) Faculty of Animal Husbandry, Universitas Brawijaya, Jl. Veterans Malang 65145 East Java Indonesia

2) National Research and Innovation Agency, Research Center for Animal Husbandry, Cibinong, Bogor, Indonesia

3) Faculty of Mathematics and Natural Science, Universitas Brawijaya, Jl. Veterans Malang 65145 East Java Indonesia

Submitted: 11 March 2023, Accepted: 3 April 2023

ABSTRACT: Artificial insemination with fresh semen is an alternative that can be done if there is a limitation of liquid nitrogen, bull, and facilities for processing liquid and frozen semen (costly). This study aimed to determine the optimal diluent formula based on CEP-3 (modified/m) and aqueous soybean extract (ASE) to support spermatozoa motility at ambient temperature storage. The research design was completely randomized, with eight treatments and six replications. The diluent formula treatments were as follows: T1 (CEP-3(m)), T2 (CEP-3(m)+10% Egg Yolk/EY), T3 (CEP-3(m)+ASE 1%), T4 (CEP-3 (m)+ASE 2%), T5 (CEP-3(m)+ASE 3%), T6 (CEP-3(m)+ASE 4%), T7 (CEP-3(m)+ASE 5%), and T8 (CEP- 3(m)+ASE 6%). The parameters were measured, i.e., the motility of spermatozoa and its kinetic parameters. Data were analyzed using ANOVA with SPSS 16 program. The result showed that the progressive motility of spermatozoa at day one was more than 50% in diluents CEP-3(m)+ASE 1-2%. Diluent CEP-3(m) and aqueous soybean extract 1-2% can support spermatozoa's motility and kinetic parameters during storage at ambient temperature, including progressive motility, motility, motility, velocity, and BCF.

Keywords: Ambient temperature; Extender; Soybean

*Corresponding Author: dian.sapo.2019@gmail.com

INTRODUCTION

Indonesia is an archipelago country with a tropical climate and various livestock genetic resources, including Ongole Crossbred cattle. The OC cattle have much superiority, so technical support is needed to increase their productivity, including reproductive technology (artificial insemination/ AI). The technology of liquid semen and frozen semen requires an expensive supporting infrastructure. These technologies' advantages are long storage (Ratnawati, Isnaini, & Susilawati, 2017; Yang, Standley, & Xu, 2018). However, the scarcity of liquid nitrogen, limitation of bull, and highly cost are obstacles in AI applications, especially outside Java Island.

One solution that can be optimized is the application of artificial insemination (AI) with fresh semen.

The advantages of AI with fresh semen are cheap and easy to do, optimal usage of bull, low dose of spermatozoa, and can be inseminated to many cows.

Appropriate semen diluents can be optimized to support spermatozoa survival at ambient temperature (long storability).

Diluents have an important role in supporting the life of spermatozoa (Coester, Sulaiman, & Riza, 2019; Susilawati, 2011).

The concept of semen diluent in ambient temperature storage has been studied by several previous studies, such as Caprogen and CEP (Verberckmoes et al., 2005).

Caudal Epididymis Plasma (CEP) diluents have been developed and refined, including CEP1, CEP2, and CEP3. The CEP diluent can be used as an ambient temperature diluent with or without adding egg yolk.

Egg yolk is very commonly used as a cryoprotectant in liquid and frozen semen.

However, egg yolk has the potential for bacterial contamination and can reduce the quality of spermatozoa (Murphy, O’Meara, Eivers, Lonergan, & Fair, 2018). Using cryoprotectants with plant sources, namely soybeans, is an alternative solution (Bousseau et al., 1998; Khalil et al., 2020;

extracted with chemical hexane or mechanically (extraction) (Bousseau et al., 1998; Layek et al., 2016; Pamungkas &

Krisnan, 2017). Several soybean extraction methods have been studied previously. The solvents used are polar solvents, including water, ethanol, etc. A combination of CEP- 2 diluent and 10% soybean extract could maintain spermatozoa motility (>40%) in Limousine cattle for up to 24 hours of cooling (Sugiarto, Susilawati, &

Wahyuningsih, 2014). Meanwhile, the use of tris diluent and 5% soybean extract could support spermatozoa motility (>40%), viability more significant than 80%, and spermatozoa abnormalities less than 20% only shortly after mixing with fresh semen of Kebumen cattle (Rezki, Sansudewa, & Ondo, 2016). This study aimed to determine the optimal CEP-based diluent formula and aqueous soybean extract to support spermatozoa motility at ambient temperature storage.

MATERIALS AND METHODS Ethical Clearance

Each stage of research related to the treatment of experimental animals has been approved by the animal ethics committee of the Indonesian Agency for Agricultural

Research and Development

(Balitbangtan/Lolitsapi/Rm/01/2022).

Place and Time of Research

The research activity was conducted for two months (January-February 2022) at the Beef Cattle Research Institute. The research material used was 1 OC bull aged 5-6 years, and the progressive motility of fresh semen was>70%. Semen was collected by using an artificial vagina. This study used a completely randomized design (CRD) with eight treatments with six replications.

The eight treatments for the semen diluents formula were: T1 (CEP-3(m)), T2 (CEP-3(m)+10% EY), T3 (CEP-3(m)+ASE 1%), T4 (CEP -3(m)+ASE 2%), T5 (CEP- 3(m)+ASE 3%), T6 (CEP-3(m)+ASE 4%), T7 (CEP-3(m)+ASE 5 %), and T8 (CEP-

Preparation Based Extender and Aqueous soybean extract

Preparation of the basic diluent CEP- 3(modified) was started by mixing all the ingredients and then stirring until homogeneous. The components of the primary diluent of CEP include KCl 0.05 mg; NaCl 0.09 mg; MgCl2(H2O)6 0.08 mg; CaCl2(H2O)2 0.04 mg; NaH2PO4 0.11 mg; NaHCO3 0.1 mg; KH2PO4 0.27 mg; Fructose 0.27 mg; citric acid 0.82 mg;

Tris 1.62 mg; streptomycin 0.1 mg;

penicillin 0.1 mg; and 100 ml sterile distilled water.

Preparation of aqueous-soybean extract begins with soaking soybeans in hot water and NaHCO3, removing the husks.

Miling soybeans with 125 ml of hot water, then filter it. The remaining dregs produced were milled with 125 ml of hot water and filtered using filtrat paper. Centrifuge the filtrated water, take the supernatant, and then filter it again (modification of (Immelda, Susilowati, & Yudaniayanti, 2019; Sartini et al., 2014)).

The basic diluent CEP-3 (m) was mixed with aqueous-soybean extract at an ambient temperature according to the percentage of treated aqueous-soybean extract. The mixture was vortexed for 1-2 minutes until the mixture was homogeneous. The diluent formula is ready to be mixed with fresh semen.

Semen processing at ambient temperature and Analysis of Spermatozoa motility by Using CASA

The diluent formula of as much as 1 ml was placed in a test tube, then 25 µl of fresh semen was added and slowly homogenized with a micropipette, covering the test tube with aluminum foil. Place the test tube into a becker glass containing water (water jacket) and store it at ambient temperature (24-27⁰C).

The CASA used in this research was SCA (Microptics v. 2.1, Spain). The

procedure of analysis of spermatozoa motility using CASA is referred to (Ratnawati, Isnaini, & Susilawati, 2018) procedure.

Parameter and Data Analysis

Parameters measured in this study including the motility of spermatozoa and their kinetic parameters, such as motility/M, progressive motility/PM, velocity curve linear/VCL, velocity average pathway/VAP, velocity straight linear/VSL, linearity/LIN, straightness/STR, wobble/WOB, beat cross frequency/BCF, amplitude lateral head/ALH, hyperactive/H (Holt, O’Brien, & Abaigar, 2007;

Ratnawati et al., 2017). Data were analyzed using ANOVA with SPSS 16.

RESULTS AND DISCUSSION Fresh Semen Quality

The initial stage of the study was to collect fresh semen from OC bull using an artificial vagina, followed by an evaluation of the quality of fresh semen. The results of observations of the quality of fresh semen are listed in Table 1.

Progressive motility and motility of spermatozoa on various diluents

The observation of progressive motility and motility of spermatozoa at ambient temperature on days 0-2 are listed in Table 2. The progressive motility of spermatozoa was decreased during storage at ambient temperature. The sharp decrease of spermatozoa motility occurred in CEP- 3(m) diluent and 10% egg yolk.

Progressive motility of spermatozoa at storage day 1 with CEP-3(m) diluents and aqueous soybean extract 1% and 2%

showed the highest value than other diluents.

Velocity of spermatozoa in Various Diluents

The observation of the velocity of spermatozoa at ambient temperature on day 0-2 are listed in Table 3.

Table 1. Fresh semen quality of Ongole Crossbred (Falchi et al.) bull

No Parameter of fresh semen quality Ongole Crossbred (Falchi et al.) bull

1 Volume 5.0 ± 2.1

2 Color creamy

3 Consistency thick

4 pH 6.4 ± 0.0

5 Mass movement (0-4) 1.5 ± 0.7

6 concentration (million/ml) 1696.5 ± 409.4

7 viability (%) 96.0 ± 2.8

8 abnormality (%) 2.0 ± 1.4

9 MP (%) 84.6 ± 11.1

10 M (%) 98.1 ± 1.2

11 VCL (µm/s) 76.7 ± 14.6

12 VSL (µm/s) 44.3 ± 12.1

13 VAP (µm/s) 64.1 ± 17.7

14 LIN (%) 57.3 ± 4.9

15 STR (%) 69.1 ± 0.1

16 WOB (%) 82.9 ± 7.3

17 ALH (µm) 2.3 ± 0.1

18 BCF (Hz) 6.7 ± 0.1

19 Hyperactive (%) 9.1 ± 2.4

Table 2. Progressive motility and motility of spermatozoa in various diluents

No Progressive motility Day of storage at ambient temperature

0 1 2

1 CEP-3(m) 82.8 ± 4.7 47.1 ± 13.2^bc 4.3 ± 4.9^ab

2 CEP-3(m)+ EY 10% 82.0 ± 5.0 6.5 ± 11.8^a 0.1 ± 0.2^a

3 CEP-3(m)+ ASE 1% 81.1 ± 7.4 55.3 ± 16.9^c 20.2 ± 16.4^b 4 CEP-3(m)+ ASE 2% 82.3 ± 6.4 56.2 ± 14.9^c 11.7 ± 11.1^ab 5 CEP-3(m)+ ASE 3% 82.6 ± 6.2 40.2 ± 16.1^bc 6.5 ± 12.8^ab 6 CEP-3(m)+ ASE 4% 84.5 ± 5.2 32.9 ± 19.2^abc 8.6 ± 18.5^ab 7 CEP-3(m)+ ASE 5% 82.9 ± 7.0 21.4 ± 6.0^ab 2.0 ± 3.1^ab 8 CEP-3(m)+ ASE 6% 78.7 ± 8.6 30.9 ± 31.5^abc 0.2 ± 0.3^a

Motility

1 CEP-3(m) 96.8 ± 2.2 70.0 ± 16.3^b 15.2 ± 11.9^ab

2 CEP-3(m)+ EY 10% 97.9 ± 1.2 35.1 ± 31.7^a 5.6 ± 8.1^a

3 CEP-3(m)+ ASE 1% 96.3 ± 2.0 77.6 ± 14.9^b 44.6 ± 20.2^b 4 CEP-3(m)+ ASE 2% 97.4 ± 1.6 75.3 ± 9.7^b 36.7 ± 22.2^ab 5 CEP-3(m)+ ASE 3% 96.7 ± 2.4 74.2 ± 12.2^b 33.9 ± 21.8^ab 6 CEP-3(m)+ ASE 4% 97.4 ± 2.3 67.8 ± 14.5^ab 25.7 ± 24.5^ab 7 CEP-3(m)+ ASE 5% 96.6 ± 1.8 65.8 ± 12.0^ab 20.1 ± 26.8^ab 8 CEP-3(m)+ ASE 6% 96.1 ± 3.5 64.5 ± 26.0^ab 8.1 ± 9.8^a Note: different superscript notations in the same column indicate a significant difference (P<0.05).

Table 3. Velocity (VCL, VSL, VAP) of spermatozoa in various diluents

No VCL Day of storage at ambient temperature

0 1 2

1 CEP-3(m) 80.8 ± 10.8 53.3 ± 6.4^bc 21.3 ± 15.8^ab

2 CEP-3(m)+ EY 10% 82.9 ± 10.2 21.6 ± 8.7^a 11.0 ± 8.7^a

3 CEP-3(m)+ ASE 1% 78.2 ± 15.9 69.8 ± 9.4^c 37.5 ± 17.0^b

4 CEP-3(m)+ ASE 2% 82.7 ± 12.5 68.2 ± 14.5^c 25.3 ± 15.9^ab 5 CEP-3(m)+ ASE 3% 85.6 ± 14.4 53.5 ± 13.3^bc 21.6 ± 12.7^ab 6 CEP-3(m)+ ASE 4% 87.0 ± 11.5 43.7 ± 12.1^ab 20.9 ± 16.3^ab 7 CEP-3(m)+ ASE 5% 79.9 ± 16.3 36.7 ± 5.7^ab 11.9 ± 10.3^a 8 CEP-3(m)+ ASE 6% 76.5 ± 12.0 41.3 ± 19.7^ab 8.9 ± 7.4^a

VSL

1 CEP-3(m) 41.7 ± 3.7 21.1 ± 4.4^c 7.5 ± 4.5^ab

2 CEP-3(m)+ EY 10% 38.5 ± 9.8 6.4 ± 5.5^a 2.2 ± 2.4^a

3 CEP-3(m)+ ASE 1% 41.4 ± 9.9 18.8 ± 6.8^bc 13.5 ± 8.2^b

4 CEP-3(m)+ ASE 2% 43.5 ± 12.6 21.2 ± 8.5^c 8.7 ± 5.2^ab

5 CEP-3(m)+ ASE 3% 47.8 ± 18.6 14.8 ± 6.5^abc 7.9 ± 3.6^ab 6 CEP-3(m)+ ASE 4% 46.8 ± 14.0 13.3 ± 5.4^abc 7.7 ± 6.5^ab

7 CEP-3(m)+ ASE 5% 46.8 ± 19.3 9.4 ± 1.7^ab 3.7 ± 3.7^a

8 CEP-3(m)+ ASE 6% 39.7 ± 10.5 12.1 ± 7.6^abc 3.9 ± 3.6^a VAP

1 CEP-3(m) 60.3 ± 5.9 32.2 ± 5.6^bcd 11.9 ± 8.5^ab

2 CEP-3(m)+ EY 10% 60.6 ± 9.8 11.4 ± 5.7^a 4.8 ± 4.1^a

3 CEP-3(m)+ ASE 1% 59.9 ± 14.8 38.1 ± 10.0^cd 21.9 ± 11.5^b 4 CEP-3(m)+ ASE 2% 64.1 ± 14.7 43.6 ± 15.8^d 13.5 ± 9.2^ab 5 CEP-3(m)+ ASE 3% 67.6 ± 18.9 29.4 ± 11.6^abcd 12.5 ± 6.3^ab 6 CEP-3(m)+ ASE 4% 68.3 ± 14.8 24.0 ± 8.7^abc 12.3 ± 9.5^ab

7 CEP-3(m)+ ASE 5% 63.2 ± 19.8 17.8 ± 3.7^ab 6.4 ± 5.7^a

8 CEP-3(m)+ ASE 6% 57.7 ± 14.2 22.0 ± 13.8^abc 5.6 ± 4.8^a Note: different superscript notations in the same column indicate a significant difference (P<0.05).

The value of VCL, VSl, and VAP of spermatozoa at ambient temperature storage decreased gradually. The decrease in the velocity of spermatozoa in the CEP- 3(m) and egg yolk 10% diluents was the largest compared to other diluents. The velocity value of spermatozoa in CEP-3(m) diluent and 2% aqueous soybean extract showed better results than other diluents.

Spermatozoa motility is one of the important parameters in determining the quality of spermatozoa because it determines the ability of spermatozoa in fertilization, viability, and structural integrity (Murphy et al., 2018). Motility is the most sensitive parameter to external conditions. Semen storage at 25⁰C or 5⁰C

for 30 minutes did not significantly affect spermatozoa viability but negatively affected spermatozoa motility (Alkali et al., 2022). Cell damage can cause spermatozoa death and be a source of toxicity for live spermatozoa (Raseona et al., 2017).

Higher levels of soybean extract decreased spermatozoa's progressive motility and velocity due to increased viscosity followed by decreased osmotic pressure. The decrease in osmotic pressure is caused by an increase in LDL concentration resulting in the precipitation of fructose and salt, which can negatively affect the fertility of spermatozoa. The debris increase also increases with the extract level (Chaudhari et al., 2017; El-

Sherbieny et al., 2022; Sisy et al., 2016;

Zhao et al., 2021).

Spermatozoa in CEP diluent and 10%

fresh egg yolk showed a sharply decreased motility on the first day of ambient temperature storage and further decreased on the second. Using fresh egg yolks potentially causes bacterial contamination rather than aqueous soybeans extract. This is appropriate with the previous study (Bousseau et al., 1998; Miguel-Jimenez et al., 2020; Silva et al., 2019), which stated that the use of egg yolks in diluents, whether fresh or not (commercial products) has a potential for bacterial contamination on a medium-high scale. Contaminant bacteria in egg yolk produce endotoxin accumulation that is toxic to spermatozoa.

The results of visual observations using CASA showed that the spermatozoa in the CEP-3(m) diluent and 10% egg yolk on the 1-2 day storage showed that there were microorganisms actively moving between the spermatozoa. Egg yolk can reduce motility, inhibit sperm metabolism and respiration, and cause difficulties during semen analysis (Mafolo et al., 2020).

Storage of spermatozoa at ambient temperature causes an increase in the rate of cell metabolism resulting in a deficit of energy sources, ATP, and accumulation of lactic acid and AAAO enzymes, so that the survival life of spermatozoa at ambient temperature (25-27°C) is short (Maulana, 2016; Raseona et al., 2017). The energy deficit causes an ATP deficit which affects the movement of the spermatozoa's tail so that the spermatozoa's motility decreases.

The high metabolism causes an increase in the by-products of spermatozoa metabolism in the form of free radicals/ROS, which are toxic to spermatozoa, including causing damage to the spermatozoa membrane that it damages DNA and proteins (Jenagrad et al., 2018; Rahimizadeh et al., 2021; Rezaei et al., 2021; Sharaf et al., 2022; Ugur et al., 2019). Metabolism by-products cause oxidative stress, so it affects decreasing motility. Damaged spermatozoa

spermatozoa cells, which causes the death of spermatozoa (Fathi et al., 2019).

The composition of the aqueous soybean extract includes protein, amino acids, vitamins, fatty acids, acetic acid, etc.

(Alkali et al., 2022). The composition in the aqueous soybean extract can support the life of spermatozoa in ambient temperature storage. Aqueous soybean extract's protein and vitamin composition is a source of energy for spermatozoa metabolism.

Amino acid components in aqueous soybean extract can act as antioxidants or ROS scavengers to control the adverse effects caused. Fatty acids play a role in restoring the phospholipid layer that ROS damaged. Aqueous soybean extract can also minimize bacteria that grow between spermatozoa based on visual observations with CASA.

CONCLUSION

The diluent formula CEP-3(m) and aqueous soybean extract 1-2% can support spermatozoa's motility and kinetic parameters during storage at ambient temperature. It was adequately to be inseminated on the first day (24 hours) of storage at ambient temperature. The use of egg yolk in the extender at ambient temperature is not recommended.

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