The Effects of Equilibration Time and Post-Thawing Temperatures in Cryopreservation of Gaga Chicken Semen

Gaga chicken is one of the local Indonesian chickens that has a unique sound that originates from South Sulawesi Province, Indonesia. Gaga chickens are bred as pet chicks and competitive chicks with a distinctive voice that resembles the laughing voice of humans. The Gaga chicken is a wealth of Indonesian livestock genetic resources that need to be protected and preserved (Indonesia Ministry of Agriculture, 2011). The diversity of local Indonesian chickens needs to be preserved to prevent them from extinction.

One way to save animals from extinction is through cryopreservation technology of gamete cells to resurrect animal species someday even if their ancestors were dead. Thelie et al. (2019) stated that chicken sperm cryopreservation is capable of fertility for 18 years. Furthermore, cryopreservation technologies of gamete cells, especially spermatozoa, can be used for Artificial Insemination (AI) in different areas that are distant from each other. However, spermatozoa are so sensitive to environmental factors during the cryopreservation process that appropriate techniques are required in their handling both before and after freezing. Moreover, according to Blesbois (2012), Spermatozoa of chicken whose filiform shape makes it more sensitive to the process of freezing and thawing.

The diluted semen needs to be balanced and temperature reduced in the process of freezing the semen (Taskin et al., 2020). Equilibration is the time the sperm adapts to the diluent used before the semen is frozen. Short equilibration can decrease the ability of sperm to adapt to the dilution, otherwise too long equilibration, spermatozoa will lose a lot of energy thereby decreasing sperm activity.

Cryopreservation causes thermal stress due to temperature changes during cooling, freezing, and thawing, and osmotic pressure due to cryoprotectant agents and crystallization in spermatozoa (Biniova et al., 2018; Khalil et al., 2018). Thawing semen quickly reduces the harmful effects of the recrystallization and hydration processes, preventing membrane damage to semen and cytoplasm. In this case, ice crystals do not form, and sperm shifts directly from freezing to liquid (El-Ahwany et al., 2018). According to Salih et al. (2021) thawing semen at a temperature of 60 °C in 5 seconds could be an efficient strategy to maintain the quality of semen chicken that is cryopreserved. In mammal species, Zenteno et al. (2023) found that the motility of bovine spermatozoa was found to be higher at a thawing temperature of 38 °C for 30 seconds compared to 36 °C for 30 seconds, while Nisa et al. (2022) found the best treatment for thawing bovine sperms to be 28 °C during 45 seconds.

The cryopreservation process involves a phase of gradual temperature decrease. The balancing process is the storage of sperm at a temperature of 5 °C before freezing. This storage aims to reduce sperm metabolic activity (Dong et al., 2008) and osmotic adaptation helps cryoprotectan enter sperm membranes (Huang et al., 2022). Equilibration time is important to note because it affects the quality of sperm post thawing. This study aims to know the effect of the equilibration time and the temperature difference in treatment thawing on the quality of the frozen semen of gaga chicken.

The study was conducted between November 2022-December 2023 at the Teaching Farm Laboratory Politeknik Pembangunan Pertanian Malang-Indonesia.

This research is a laboratory experiment. The samples of fresh semen came from 10 male chickens were the study animals with different body condition category. The criterion of fresh semen used in this study must have a sperm motility of ≥70% to be processed into frozen semen (BSN, 2021).

This study used a complete random design of factorial patterns with factor I being the equilibration time and factor II being the different thawing conditions.

Factor I: P0 = 0 hour (without equilibration)

P1 = 1 hour equilibration

P2 = 2 hours equilibration

Factor II: T1 = 28 °C thawing temperature for 45 seconds

T2 = 38 °C thawing temperature for 30 seconds

T3 = 60 °C thawing temperature for 5 seconds

In determining the number of samples using the Federer formula is: (n-1) (t-1) ≥ 15 where (t) is the count of treatment groups, and (n) is number of repetitions.

The extender used was 90 % Ringer Lactate (PT Satoria Aneka Industri) and 10% egg yolk (Junaedi et al., 2016). It was centrifuged for 15 minutes at a speed of 3000 rpm. The supernatant was taken and added 7% DMSO or dimethyl sulfoxide (Merck, KgaA, 64721 Darmstadt, Germany), 1000 IU/ml penicillin (PT Meiji, Indonesia) and 1 mg/ml streptomycin (PT Meiji, Indonesia). The pH of the diluent was adjusted with the addition of tris (hydroxymethyl) aminomethane (Merck, KgaA, 64721 Darmstadt, Germany) up to pH 7.4.

The study used semen from 10 male Gaga chickens 10 months old, healthy, normal reproductive organs. Semen was collected using massage methods on the back of the chicken. At the time of the sheltering, the hands form a 45° angle with the backbone (os vertebrae lumbales et sacrales) of chicks. The semen that comes out is stored in a petri cup and avoided contamination by dirt. Gaga chickens were kept in individual cages equipped with feed and drinking water. The daily feed commercial was 100 grams. Drinking water was given ad libitum. Semen was divided into 9 tubes and each diluted according to treatment with a sperm concentration of 50x106 in 0.25 ml of mini straw (IMV, France).

The straw was then equilibrated at a temperature of 5 oC based on treatment for P0=0 hours (without equilibration), P1= 1 hours of equilibration and P2=2 hours of equilibration. It was pre-freezing for 10 minutes by placing the straw on a liquid nitrogen surface a distance of 3 cm (Madeddu et al., 2016), and then put into a liquid nitrogen container (-196 °C) for 24 hours (Khairuddin et al., 2019).

Macroscopically of Semen Evaluation adopting Arifiantini (2012). The macroscopic evaluation covers the volume of semen measured by looking at the scale on the semen housing tube. The colour is seen visually, fresh semen can be milk white, cream or yellowish. Acidity (pH) is measured using pH indicator paper (range 6, 4-8, Merck Germany). The consistency of the semen is assessed by bending the container tube and returning it to its original position. Consistency is seen based on the speed of semen returning to the base of the tube. The consistency of the semen ranges from low to medium.

The evaluation of frozen semen (more than 24 hours) after thawing was done by placing frozen semen in a water bath based on temperature treatment T1=28 °C, T2= 37 °C, T3=60 °C for 5 seconds. Percentage of sperm motility, percentage of sperm viability, PMI, and acrosome integrity were evaluated after thawing.

A total of 10 μL of frozen semen after thawing then was observed using a binocular microscope (Olympus CX-23, Japan) with a 400X magnification of microscope, and observations were made of the number of progressive motile spermatozoa from one field of view and expressed in percentages (Khalek et al., 2018). Under a light microscope, Spermatozoa were assessed in each smear in at least five distinct fields. Sperm were classified as motile or nonmotile individually. From a single field of view, observations were made about the total number of progressing motile spermatozoa and their velocity, which were then expressed as a percentage.

The percentage of sperm viability was done by the eosin-nigrosin (Merck, KgaA, 64721 Darmstadt, Germany) colouring method. One drop of diluted frozen semen after thawing, placed on the object glass, then added with one drop of eosin-nigrosin (Merck, KgaA,64721 Darmstadt, Germany) dye (1:3) and then homogenized. Then the preparation is made by pressing and pushing the object glass to form a 45° angle and dried. Then observe with a magnification of 400X- microscope in ten fields of view or 200 cells of sperm. The dead sperm will absorb red matter because the permeability of the cell wall has been weakened while the living sperm would look transparent (Silyukova et al., 2022).

Observation of PMI using hypoosmotic swelling test techniques. 10 μL semen diluted in hypoosmotic solution (0.9 g of fructose and 0.49 g of sodium citrate (Merck, KgaA, 64721 Darmstadt, Germany) dissolved in 100 μL of distillation water) kept at a temperature of 37 °C for 30 minutes, 200 of cell spermatozoa were evaluated which indication for intact was swelling in the tail of spermatozoa, and if not swelling in the tail of spermatozoa so damaged plasma membrane (Mehdipour et al., 2018; Najafi et al., 2022).

Observation of intact acrosome using colouring solution of Coomassie Brilliant Blue (CBB) (Merck, KgaA,64721 Darmstadt, Germany). The semen was placed on a glass of the object and then dried and scrubbed for 30 minutes in a 5% formalin solution at a temperature of 37 °C, rinsed with distilled water and air-dried. The dyeing procedure is based on Silyukova et al. (2022) that semen was dried for 5 minutes in a CBB solution (0.25% Coomassie Brilliant Blue R 250 in a solution of 10% glacial acetate acid and 25% methanol) and then rinsed with distillation water and dried with air. Observations of acrosome integrity are done at 5-6 fields of view in a 100x magnification microscope. The whole spermatozoa acrosome was concentrated blue, while the unintegrated acrosome was less coloured.

The all data were analyzed using a two-way analysis of variance (ANOVA), if the F-value was significant (P<0.05) then it was continued with the Duncan multiple range test. Statistical analysis using the SPSS application version 27.0, and presented as mean ± standard error (SE).

The quality of fresh semen gaga chicken in this study included (a) the evaluation of macroscopic among others: volume, colour, pH, and smell; (b) microscopic among others: mass motility, individual motility, viability, concentration, and abnormality. The characteristic of chicken fresh semen quality in the study was good, with an average volume 0.18± 0.06 mL, and pH 8.10±0.16. the colour was milky white to cream, has a thick consistency, has average concentration 3.69±1.02, mass movement +++, motility 83.75±2.50, viability 98.20±0.33, PMI 97.25±3.80, and abnormality 12.67±1.31. The results of the macroscopic and microscopic evaluation of fresh gaga chicken semen showed good quality for freezing.

Motility of chicken spermatozoa experienced a significant decrease after thawing (Khaeruddin et al., 2022). The differences in the equilibrium time caused by the motility of chicken spermatozoa after freezing are also different (Zhong et al., 2023).

The results of analysis of variance show that the treatment of different thawing and equilibrium times have no significant influence (P>0.05) on the motility of gaga chicken semen (Table 1). The motility of the gaga chicken semen after freezing with different equilibration and different thawing shows ranging from 33.00±1.13 to 35.80±1.03. The study showed that there were no significant differences between treatments (P>0.05). Lower than the motility in previous research reports, that was chicken frozen semen motility after thawing has 38.6% using Beltsville Poultry Semen Extender (BPSE) with hyaluronic acid added (Lotfi et al., 2017), 38% in Bangkok chicken freezing semen using BPSE with DMA cryoprotectant (Laura et al., 2017), 37.22% in chicken semen with egg yellow milk ringer with DMSO cryoprotectant (Junaedi et al., 2016), and 35.8% using L-carnitine (Fattah et al., 2017). The results obtained in this study are higher than Shahverdi et al. (2015) which has 22.7% using Beltsville extender and Pranay et al. (2018) who got 27.73%-34,55% using the N-methyl acetate added. That was in line with the Milanda and Fattah (2022) which stated that there was no difference in the time of thawing against the motility of the frozen semen of domestic chicken ((P>0.05).

Table 1: Percentage motility of sperm Gaga chicken in post thawing with treatment equilibration time and treatment thawing.

Viability of sperm gaga chicken was observed with eosin nigrosin colouring will remain bright, whereas dead sperms will absorb the eosin nigrosin colour. That was because the Na+ pump in the living sperm works well whereas in the dead sperm Na+ pumps do not work (Figure 1).

Average percentage of viability of sperm Gaga chicken after freezing is presented in Table 2. Semen of Gaga Chicken after thawing with different equilibration time and thawing lengths shows ranges between 45.68±3.00 to 50.85±1.85. The study showed that there were no significant differences between treatments (P>0.05). That was because it protects sperm plasma membranes against the internal and external electrolyte-electrolyte balance of spermatozoa, so that the sperm metabolism process was uninterrupted so that it can maintain sperm survival. This result was higher when compared to Mosca et al. (2016) in Lohman chickens, for DMA (Dimethyl Acetamide) produced live spermatozoa which were 46.9%, respectively, and Setioko et al. (2002) on the duck semen using DMA and DMF for cryoprotectant produced viability of sperm post thawing that were 48.04 and 39.88%, respectively.

Table 2: Percentage viability of sperm Gaga chicken in post thawing with treatment equilibration time and treatment thawing.

Table 3: Percentage PMI of sperm Gaga chicken in post thawing with treatment equilibration time and treatment thawing.

Average percentage of PMI of sperm Gaga chicken after freezing was presented in Table 3. Chicken Gaga spermatozoa after frozen with different balancing time treatments show best treatment on treatment P2 averages 51.81±2.97 (P<0.01) compared to P0 43.29±2.91 % and P1 45.46±2.60. While different thawing temperature treatments indicate difference between treatments. On treatment T2 and T3 were best 48.42±2.88 and 49.06±2.67(P<0,01) and last on T1 43.08±2.93 (P>0.01).

Semen freezing can result in physical stress, often called cold shock, which affects structural and biochemical damage, thereby affecting cell function and ultimately causing cell death (Khan et al. 2021). Cold shock causes changes in the phospholipids that make up the plasma membrane during the transition phase from the liquid to the freezing phase (Ghetler et al., 2005), changes in plasma lipid chain and protein membrane arrangements that can result in leakage or decreased selectivity (Dziekońska and Strzeżek, 2011). The plasma membrane will lose its selective permeability and affect many cellular components such as lipids, proteins and ions it releases (Blesbois, 2012). When there is damage to the plasma membrane due to cold shock, the glucose-6-phosphate dehydrogenase enzyme first leaves the cytoplasm, which generally leads to a decrease in intra-cellular ATP concentration (Lemma, 2011). There are also leaks of enzymes that play a role in metabolic processes such as aspartate aminotransferase (AspAT), the ATPase-linked sodium-potassium pump enzyme (Na+/K+-ATPase) (Arifiantini and Purwantara, 2010), Cold shock also predicts the dislocation of plasma membrane proteins such as glucose transporter groups, which have a role to play in the transport of hexose through plasmatic membranes (Kokk et al., 2005).

DMSO is a mixture of organosulfur with the chemical formula [Me2SO, (CH3)2SO] and has a small molecular weight, hence the DMSO known as an intracellular cryoprotectant. The addition of DMSO aims to maintain membrane integrity and increase the osmotic potential of the medium so that the fluid inside the cell flows out and dehydration occurs (Erol et al., 2021). The ability of the cryoprotectant to protect against cell membranes was an indication of a functioning interaction between the cryoprotectant and the cell membrane. This interaction can reduce damage to the membrane of the cell when the condition changes from relatively liquid to relatively dense structure and also when it returns to a relatively fluid structure during the thawing process (Mehaisen et al., 2022).

The PMI is an absolute thing that spermatozoa must have. The plasma membrane serves as the first protection of cells from the external environment that can damage cells. Spermatozoa that suffer from plasmatic membrane damage are characterized by a straight tail, whereas spermatozoa with an PMI are characterised by a circled or swelling in the tail of sperm (Figure 2).

Table 4: Percentage acrosome integrity of sperm Gaga chicken in post thawing with treatment equilibration time and treatment thawing.

Average percentage of integrity of acrosome in the Gaga chicken semen after freezing was presented in Table 4. Gaga spermatozoa after freezing with different equilibration time treatments did not show any significant difference between treatments (P>0.01), the largest of P1 45.91±2.38, P2 44.90±2.75, P0 42.86±2.27. While thawing treatments showed a marked difference between treatment (P<0.01) T3 of 52.78±2.51, T2 45.15±2.93, and T1 of 35.74±1.96.

CB stain was commonly used in protein identification. Coomassie Blue is able to bind to membrane proteins, by binding to glycoprotein (Silyukova et al., 2022). The spermatozoa acrosome membrane consists of the Inner Acrosome Membrane (IAM) and the Outer Acrosome Membrane (OAM) which serves as the protector of the sperm of acrosome that will be coloured (Khawar et al., 2019). When part of the acrosome of the spermatozoa cells is damaged or imperfect, sperm cells are unable to absorb colour properly and the colouring results will fade (Figure 3). An irregularity in the head can also be used as one of the indications that the plasma membrane has suffered damage as well as potential damage to the acrosome.

In conclusion, there is no difference of influence between the long equilibration and the temperature difference of the thawing on the quality of the frozen chicken semen. this study showed that all treatment gives quite good results on motility and viability of sperm Gaga chicken. A two-hour equilibration treatment was capable of preserving plasma membrane integrity and acrosome integrity at a 38°C thawing temperature for 30 seconds or a 60 °C thawing temperature of 5 seconds. Further research was needed on the successful implementation of artificial insemination.

The authors would like to thank the Ministry of Research Technology and Higher Education of the Republic of Indonesia for having granted this project through Hibah Professor University of Brawijaya year 2023 with number granted: 2044.2/UN10.F05/PN/2023.

This is the first study to report the effects of equilibration time and post thawing temperatures in the cryopreservation of Gaga chicken which is a local Indonesian chicken that needs to be protected and preserved.

SW, AAA, KH, HP and ARIP were conceptualized and designed the experiment, literature search, and wrote manuscript drafts. SW and AAA edited and revised the manuscript. KH, HP and ARIP helped with interpretation of analytical statistic. All authors were critically read, reviewed and approved the final manuscript.

All procedures in this study have obtained approval from Research Ethic Commission, University of Brawijaya for the use of animals with certificate number: 096-KEP-UB-2023.

The authors have declared no conflict of interests.