Effect of Cysteamine on the Freezing of Buck Semen

Address for Correspondence: Asiye İzem SANDAL • E-mail: izem@istanbul.edu.tr

Received Date: 18 October 2019 • Accepted Date: 10 February 2020 • DOI: 10.5152/actavet.2020.19029 Available online at actavet.org

Abstract

This study investigated the effect of cysteamine added to an extender at different doses (5 and 10 mM) on the freezing of Saanen buck semen containing samples with seminal plas-ma or those in which the seminal plasplas-ma was removed. Af-ter the examinations, the ejaculates were pooled. The semen was divided into two equal volumes. The seminal plasma of one group was not removed (Group A), whereas the seminal plasma of the other volume was removed via centrifugation (Group B). Each group was again divided into three equal volumes. Therefore, a total of six groups were created. Sub-sequent to the equilibration process, diluted semen samples were packaged in 0.25 mL straws, frozen at −110°C, and

sto-red at −196°C. Frozen semen samples were thawed in a water bath for 30 s at 37°C. This procedure was repeated seven ti-mes (n=7). In the equilibration stage, 10 mM cysteamine was found to damage the spermatozoa motility regardless of the presence of seminal plasma (p<0.001). After thawing, no sta-tistically significant difference was observed in all the groups. In this study, it was concluded that 10 mM cysteamine dama-ges spermatozoa motility before freezing and the presence of seminal plasma and cysteamine concentrations after thawing had no effect on spermatological properties.

Keywords: Antioxidant, buck semen, cysteamine, freezing

Effect of Cysteamine on the Freezing of Buck Semen

Ezgi ERTÜRK

_{, Hatice ŞENLİKÇİ}

_{, Asiye İzem SANDAL}

_{, Özen Banu ÖZDAŞ}

_{, Kemal AK}

1_{Veterinarian, İzmir, Turkey}

2_{Department of Reproduction and Artifical Insemination, İstanbul University-Cerrahpaşa, Faculty of Veterinary Medicine, İstanbul, Turkey}

Cite this article as: Ertürk, E., Şenlikçi, H., Sandal, A.İ., Özdaş, Ö.B., Ak, K., 2020. Effect of Cysteamine on the Freezing of Buck Semen. Acta Vet Eurasia 2020; 46: 63-67.

ORCID IDs of the authors: E.E. 0000-0001-9079-9212; H.Ş. 0000-0002-9568-2088; A.İ.S. 0000-0002-4952-7861; Ö.B.Ö. 0000-0002-6867-5915; K.A. 0000-0002-4053-9655.

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

Introduction

Breeds with high meat and milk yield characteristics can be ob-tained via genetic selection and breeding studies on livestock. In Turkey, milk and meat yields from sheep and goat breeds are very low. Effective genetic improvement can be achieved in breeding programs by performing artificial insemination using semen from superior-quality goats. However, there are factors limiting the application of artificial insemination in small rumi-nates. During the freezing of semen samples taken from rams and bucks, problems occur in the morphological structure of the spermatozoa, including acrosome degenerations, DNA damages, and particularly reduced motility (Gordon, 2005). One of the most important handicaps encountered during the freezing of buck semen is the interaction of the bulbourethral gland enzyme contained in the seminal plasma of bucks with

egg yolk and milk powder in diluents and sperm agglutination. Researchers separated the semen from its seminal plasma be-fore the freezing process to resolve this problem (Cabrera et al., 2005; Kozdrowski et al., 2007). Furthermore, there are studies involving the freezing of buck semen aiming at preventing oxidative stress by using various antioxidant agents (Alçay et al., 2016; Seifi-Jamadi et al., 2017). In the freezing process of semen, it is very important to know the properties of mamma-lian semen, the chemical structure of extenders, dilution rate, and freezing–thawing rate (Ak et al., 2010; Sarıözkan, 2008). In order to increase their success rates, energy and antioxidant agents (ergothioneine, cysteamine, cystine, cysteine, taurine, and hypotaurine) are used in various extenders (Kulaksız and Daşkın, 2009; Tuncer et al., 2010). Antioxidant agents are known to prevent lipid peroxidation in mammalian semen, protect se-men from the negative effects of reactive oxygen species (ROS),

and positively increase the motility and viability of semen after freezing–thawing processes (Tuncer et al., 2010). The existing antioxidant systems in spermatozoa are insufficient to protect them from the effects of ROS. This is due to the small volume of cytoplasm in the cell and low concentration of enzyme release. Furthermore, antioxidant systems of seminal plasma may be damaged during the application of assisted reproductive tech-niques (Sapanidou et al., 2015). Sperm cells can get damaged by ROS during freezing–thawing processes. It is difficult to pre-vent semen from getting damaged since the aerobic metabo-lism of semen can also produce ROS. The aim of this study was to determine the effect of cysteamine-an antioxidant agent endogenously produced by the body-on the spermatological characteristics of buck semen following freezing–thawing pro-cesses.

Materials and Methods

Animal care and feeding

In this study, semen samples were obtained from five fertile Saanen bucks (age: 1–3 years). Care and feeding of the bucks were carried out under standard breeding conditions at the an-imal shelters of the İstanbul University-Cerrahpaşa, Faculty of Veterinary Medicine Department of Reproduction and Artificial Insemination.

Semen collection

The semen samples were taken twice a week using an elec-tro-ejaculator during the mating season (between Septem-ber and January). After the bucks were accustomed to giving semen samples and the preliminary studies were complet-ed, experiments using statistical data were performed and repeated seven times (n=7). The semen was collected twice a week from each buck by the electro-ejaculation method; during this time, the bucks were sedated with 0.22 mg/kg in-tramuscular injections of xylazine (Ege Vet, Izmir, Turkey) and 1.10 mg/kg flunixin meglumine (Intervet, Istanbul, Turkey). Semen was obtained with the aid of small electrical currents using an electro-ejaculator device (Ruakura, MK IV Ram Probe; Alfred Cox, Surrey, UK). This process was repeated 10 times. During the semen collection procedure, a pre-lubricated rec-tal probe was inserted about 10 cm into the rectum of the sedated buck, laying on its side. The semen was collected into sterile tubes heated to 35°C at specific time intervals and in sets; a total of 10 electrical stimulations were applied. All the procedures on animals during this study were carried out according to the approval of the Local Ethics Committee for Animal Experiments of Istanbul University (approval number: 2013/90).

Evaluation of spermatological characteristics

The semen samples were delivered to the laboratory within a short time. Specimens that were found to be macroscopically appropriate (volume, color, odor, and consistency) were placed in a water bath at 26°C.

Mass activity

A 5 μL undiluted semen sample put on a slide was assessed un-der a light microscope with a hot stage without being covered with a cover glass under 10× magnification. The +4 unit was used to evaluate the mass activity, and samples with a score of at least +3 were included in this study.

Motility

Samples with a semen/extender ratio of 1:3 were examined under a warming-plate phase-contrast microscope after they were transferred to the slide and covered with a cover glass. Spermatozoa motility was evaluated in percentage (%) by con-sidering forward-moving spermatozoa. Samples with motility of 80% and over were used in this study.

Spermatozoa density

The number of spermatozoa per unit volume (mL) was deter-mined using a hemocytometer. Semen samples with

sperma-tozoa density of 1.5×109 _{and below were excluded from this}

study.

Morphological examinations

Here, 5 μL samples taken from fresh semen in order to un-dertake morphological examination were determined in a Hancock solution. The morphology of the spermatozoa was assessed under phase-contrast microscopy under 100× magni-fication (Hancock, 1952).

Viability ratio (percentage of live/dead cells)

The viability ratio was examined with the help of the eosin vital dye (Öztürkler et al., 2001). A smear slide was prepared and a total of 200 spermatozoa were counted in the dry smear at 40× magnification. Cells that were not stained with the vi-tal dye and appeared white in the nigrosine background dye were considered to be viable and were recorded in percent-age (%).

Hypoosmotic swelling test (HOST)

The HOST was performed to determine the membrane integrity of the spermatozoa. For this purpose, the HOST solution at the osmolarity of 100 mOsm/kg (0.9% fructose weight/volume and 0.49% sodium citrate weight/volume) was prepared. After the samples were incubated for 1 h at 37°C, 200 cells were count-ed under 100× magnification. Spermatozoa showing swelling and curling of the tail were considered to be viable and showed membrane integrity (Tuncer et al., 2010).

Dilution and Freezing of Buck Semen Creation of groups

The ejaculate was divided into two equal volumes following the pooling process. No centrifugation was performed for the first volume (Group A), whereas the second volume was centrifuged at 2000 g for 5 min (Purdy, 2006) and the seminal plasma was removed (Group B). The dilution rate was

straw with a Tris-fructose-citric acid (17% egg yolk) extender. Each volume was again divided into 3 equal volumes: Groups A0 and B0 without cysteamine, Groups A5 and B5 containing 5 mM cysteamine, and Groups A10 and B10 containing 10 mM cysteamine. Semen samples from these groups were cooled to 5°C by decreasing the temperature at the rate of 0.3°C per min. Cooling was performed by means of a Bio-Cool® instru-ment (BC-III-40; SP Industries, New York, USA) at the rate of 0.3°C per min (Ak et al., 2010). Samples from Groups A and B were subjected to glycerolization at 5°C, and the final glycer-ol concentration was 5%. Glycerglycer-olization was performed in 4 volumes (10, 20, 30, and 40%) at 8-min intervals, and the di-luted semen samples were left for equilibration for 45 min. At

the end of the equilibration process, diluted semen were put in 0.25 mL straws and their ends were sealed with polyvinyl al-cohol. Straws placed on straw combs with the help of a straw ramp were frozen in liquid nitrogen vapor for 10 min at 110°C. After freezing, all the straws were stored in a liquid nitrogen tank at −196°C until the day of examination. After storing in liquid nitrogen for at least three days, the spermatological ex-aminations were completed.

Thawing and examination of buck semen

The frozen semen in the study groups were thawed for 30 s in a water bath at 37°C. Then, the thawed samples were transferred to tubes and placed on a warming plate (at 37°C) for examina-tion.

Statistical analysis

Statistical analyses were performed by using the General Linear Model procedure in Statistical Package for the Social Sciences (SPSS Inc.; Chicago, IL, USA) version 13.0 software to determine the effect of cysteamine dose added to the extender on buck semen after the freezing procedure and whether the seminal plasma was removed or not. In this statistical model, A/B status (Group A: seminal plasma was removed; Group B: seminal plas-ma was not removed), cysteamine dose added to the extender (0, 5, and 10 mM) (Table 1), and A/B status×dosage interaction were included.

Results

The spermatological characteristics of the groups after the equilibration and thawing processes are summarized in Table 1. After equilibration, all the cysteamine concentrations (0, 5, and 10 mM) in Groups A and B were compared with each other: the motility values of Groups A0 and B0 without cysteamine were found to be significantly lower (p<0.001). Motility, morphology, viability, and HOST values after equilibration and thawing were found to be similar for all the groups (Tables 2 and 3).

Table 1. Spermatological parameters after equilibration and thawing (%)

Parameters A/B Status Cysteamine Dosage (mM) Significance (p value)

A B 0 5 10 SE A/B Dosage ABxDosage

Motility % After equilibration 71.19a _66.91a _75.00a _73.21a _58.93b _{1.736 0.225 <0.001 0.330} After thawing 43.33 42.38 43.93 45.36 39.29 2.056 0.818 0.460 0.710 Viability % After equilibration 65.91 62.81 65.93 65.79 59.86 1.833 0.571 0.316 0.720 After thawing 37.52 40.95 39.14 39.36 39.21 1.688 0.317 0.999 0.776 Acrosome Defects % After equilibration 2.29 1.95 1.86 1.93 2.57 0.127 0.199 0.054 0.054 After thawing 2.95 3.05 2.57 3.14 3.29 0.251 0.851 0.477 0.104 Total Aabnormlity % After equilibration 15.33 15.76 15.71 14.79 16.14 0.717 0.767 0.734 0.656 After thawing 23.57 25.05 24.64 24.50 23.79 0.854 0.393 0.908 0.908 Host% After thawing 38.62 39.33 40.50 37.50 38.93 2.196 0.872 0.856 0.683

A: Groups containing seminal plasma, B: Seminal plasma-free groups. a,b_{The difference between values that do not have common letters on the same line is significant}

(p<0.001). SE: standard error

Table 2. Spermatological parameters after equilibration

Dosage of Group A Group B cysteamine (with seminal (seminal Parameters (mM) plasma) plasma free) Motility (%) 0 77.14±4.253 72.86±4.253 5 72.14±4.253 74.29±4.253 10 64.29±4.253 53.57±4.253 Viability (%) 0 66.14±4.49 65.71±4.49 5 65.57±4.49 66.00±4.49 10 63.00±4.49 56.71±4.49 Acrosome defects (%) 0 2.29±0.31 1.43±0.31 5 2.29±0.31 1.57±0.31 10 2.29±0.31 2.86±0.31 Total abnormality (%) 0 25.71±0.09 25.57±0.09 5 23.43±0.09 24.00±0.09 10 23.57±0.09 24.00±0.09 MV±SE, p>0.05 Mean Value (MV) Standard Error (SE)

Discussion

In this study, the effects of cysteamine added to the Tris ex-tender at different concentrations on the spermatological characteristics of the semen with and without seminal plasma after equilibration and freezing processes were investigated. Regardless of the seminal plasma factor, the most prominent results in this study were evident in the motility values after equilibration. The motility of the samples with a cysteamine concentration of 10 mM was found to be lower than those for the other two groups (without cysteamine and with 5 mM cys-teamine) (p<0.001). Samples without cysteamine and samples containing 5 mM cysteamine can be used in the Tris extender; however, an intense cysteamine concentration (such as 10 mM) is harmful to spermatozoa motility. Although the same situa-tion was found to be statistically insignificant, this trend con-tinued after the thawing process (Table 1). This harmful effect did not change according to the presence of seminal plasma. In several studies, the addition of 5 mM cysteamine has been reported to have beneficial effects (Alçay et al., 2016; Bucak et al., 2009; Kulaksız and Daşkın, 2009). In the literature, there are no studies on semen extenders containing cysteamine at the specified concentrations. Although the cause is not fully understood, the harmful effect of 10 mM cysteamine can be attributed to seminal plasma, extender contents, and chemical interaction of cysteamine (Pellicer-Rubio et al., 1997). Semen cells may be influenced by different factors such as semen ex-tender contents, ratios, and animal breed. In a study by Bucak

et al. (2009), semen samples were collected from Angora bucks, which were diluted with a Tris-based extender containing the antioxidants of hypotaurine (5 mM) and cysteamine (5 mM), an amino acid solution (13%), and an extender containing no antioxidants (control). In the same study, antioxidants were re-ported to cause a significant increase in spermatozoa motility, morphology, and functional membrane integrity evaluated with HOST and morphological examinations, without influenc-ing ROS formation after freezinfluenc-ing–thawinfluenc-ing. In the present study, 5 mM cysteamine was found to have neither a positive nor a negative impact. The data obtained from our study do not sup-port the findings from other studies, and this may be attributed to physical and chemical reasons. Although the motility rates of Group B10 without seminal plasma among the groups in which 10 mM cysteamine is added before and after freezing were found to be statistically insignificant, it was seen to be remark-ably lower than those in the other groups (Tables 2 and 3). This decrease can be attributed to the lower number of repetitions and/or centrifugation procedures (Naing et al., 2011). Cabrera et al. (2005) reported that the removal of seminal plasma could increase the motility rates in the freezing of buck semen. In the study of Tabarez et al. (2017), similar motility rates were found in buck semen with and without seminal plasma after thawing. In conclusion, it has been found that 10 mM cysteamine dam-ages spermatozoa motility before freezing and the presence of seminal plasma and cysteamine concentrations after thawing had no impact on the spermatological properties. Further stud-ies are necessary to determine the effect of cysteamine on the freezing of buck semen.

Ethics Committee Approval: Ethics committee approval was received from the Animal Experiments Local Ethics Committee of Istanbul Uni-versity (2013/90).

Peer-review: Externally peer-reviewed.

Author Contributions: Concept – K.A.; Design – K.A., E.E.; Supervision – K.A., Ö.B.Ö.; Resources - K.A., Ö.B.Ö.; Materials – E.E., H.Ş.; Data Collec-tion and/or Processing - E.E., H.Ş., A.İ.S.; Analysis and/or InterpretaCollec-tion - E.E., H.Ş., A.İ.S.; Literature Search – E.E., K.A.; Writing Manuscript –Ö.B.Ö., A.İ.S., K.A.; Critical Review – A.İ.S., Ö.B.Ö., K.A.

Conflict of Interest: The authors have no conflicts of interest to de-clare.

Financial Disclosure: The authors declared that this study has received no financial support.

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