Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido: 19/12/2024 Aceptado: 29/03/2025 Publicado: 02/05/2025 hps://doi.org/10.52973/rcfcv-e35595 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 6 Comparison of the Preservave Eﬀects of Trehalose, Iodixanol and EDTA in the Cryopreservaon of Ram Sperm Collected by Electroejaculaon Comparación De los Efectos Protectores de la Trehalosa, El iodixanol y EDTA en la Criopreservación De Semen De Carnero Recolectado Mediante Electroeyaculación Mehmet Ferit Özmen 1* , Ramazan Arıcı 2 , Ümüt Cirit 3 , Kemal Ak 2 ¹ Department of Reproducon and ArﬁcialInseminaon, Faculty of Veterinary Medicine, Dicle University, Diyarbakir,Turkey. ² Department of Reproducon and ArﬁcialInseminaon, Faculty of Veterinary Medicine, Istanbul University-Cerrahpaşa, Istanbul. ³ Department of Reproducon and Arﬁcial Inseminaon, Faculty of Ceyhan Veterinary Medicine, Cukurova University, Adana,Turkey. *Corresponding authors: ferit-ozmen@hotmail.com ABSTRACT Improving spermatological quality in frozen ram semen is very important for the development and disseminaon of arﬁcial inseminaon in sheep. For this purpose, researchers have added many substances in various proporons to sperm extenders and invesgated the eﬀects of these substances. The presented study compares the protecve eﬀects of adding Trehalose, Iodixanol, and EDTA, both individually and in diﬀerent combinaons, to the diluent for freezing ram sperm. The study concluded that; (1) trehalose, iodixanol or EDTA signiﬁcantly contribute to the preservaon of the funconal and morphological integrity of spermatozoa, (2) while the addion of iodixanol or EDTA alone to the basic extender failed to increase total and progressive molity aﬅer thawing, when used together they produced a synergisc eﬀect and signiﬁcantly increased total molity, (3) the use of trehalose, iodixanol and EDTA together at appropriate concentraons or the addion of 5% iodixanol or 1.5 g/L EDTA alone to the extender increased spermatozoon persistence aﬅer thawing. Key words: Ram semen; EDTA; lodixanol; trehalose RESUMEN Mejorar la calidad espermatológica del semen congelado de carnero es crucial para el desarrollo y la difusión de la inseminación arﬁcial en ovinos. Para ello, los invesgadores han añadido muchas sustancias en diferentes proporciones a los extensores de esperma y han invesgado los efectos de estas sustancias. El estudio presentado compara los efectos protectores de añadir Trehalosa, Iodixanol y/o EDTA, tanto individualmente como en diferentes combinaciones al diluyente, para congelar el semen de carnero. El estudio concluyó que: (1) la trehalosa, el iodixanol o el EDTA contribuyen signiﬁcavamente a la preservación de la integridad funcional morfológica de los espermatozoides, (2) mientras que, la adición de iodixanol o EDTA por sí sola al diluyente básico no aumentó la molidad total progresiva después de la descongelación, cuando se usaron juntos produjeron un efecto sinérgico y aumentaron signiﬁcavamente la molidad total, (3) el uso de trehalosa, iodixanol y EDTA juntos en concentraciones apropiadas o la adición de 5% de iodixanol o 1.5 g/L de EDTA por sí sola al diluyente aumentó la persistencia de los espermatozoides después de la descongelación. Palabras clave: Semen de carnero; EDTA; iodixanol; trehalosa

Eﬀects of Trehalose İodixanol and EDTA on Ram Semen / Özmen et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTİON It is well-documented that sperm freezing induces biochemical, ultrastructural and funconal alteraons in spermatozoa [1]. Because the plasma and acrosome membranes of spermatozoa are sensive to freezing, the permeability of the cell membrane increases, which may negavely impact sperm molity, morphology, and chroman integrity [2 ,[3]. Furthermore, hyper-oxidaon and the generaon of reacve oxygen species take place, potenally leading to damage to the mitochondrial sheath and tail axoneme. Numerous scienﬁc studies have been conducted to reduce these adverse eﬀects and enhance ferlizaon capacity in frozen-thawed ram semen. To achieve this, permeable cryoprotectants (glycerol, dimethyl sulfoxide), non-permeable cryoprotectants (egg yolk, skimmed milk), sugars (lactose, raﬃnose, trehalose, glucose), salts (sodium citrate, citric acid), and anoxidants (amino acids, enzymes, vitamins), herbal extracts (ginkgo biloba, capsaicin) were incorporated into the sperm diluent and evaluated [4 ,5, 6 , 7 , 8]. Trehalose, a disaccharide, protects spermatozoa by creang a hypertonic environment during the glycerolizaon and equilibraon phases and by inﬂuencing intracellular crystallizaon aﬅer dehydraon. This cryoprotecve eﬀect is explained by trehalose’s ability to regulate membrane ﬂuidity [9 , 10]. It was also reported that trehalose could change the protein structure of ram sperm during cryopreservaon and have anoxidant eﬀect on frozen spermatozoa, parcipaon in glycolysis and increase the tolerance of spermatozoa to various stress factors [11]. Ethylenediaminetetraacec acid (EDTA) reduces the freezing temperature of disaccharide soluons [1]. Aisen et al. [12] indicated that combining trehalose and EDTA in a tris extender provided beer protecon against the eﬀects of freezing on sperm compared to using trehalose alone. Originally developed as an x-ray contrast agent, iodixanol is now commonly used as a centrifugaon medium for isolang live cells due to its non-toxic nature to cells [13 , 14]. The successful results in studies using iodixanol for centrifugaon in sperm freezing suggest that the minimal residual iodixanol, which remains with the pellet at the boom aﬅer centrifugaon and cannot be aspirated, may have provided a cryoprotecve eﬀect. In order to invesgate this hypothesis, Saragusty et al. [15] tried iodixanol for the ﬁrst me in the freezing of bull semen and obtained highly posive results. Although the exact mechanism of acon is not yet completely understood, the study revealed that the addion of iodixanol to the bull sperm extender raised the glass transion temperature and altered the structure of ice crystals formed during freezing. This modiﬁcaon transformed the crystals into a form that would either not damage or cause less damage to spermatozoa [15]. No other similar study was found examining the eﬀects of using iodixanol, EDTA and trehalose together or separately on frozen ram semen. However, there are studies invesgang the eﬀects of using two of these three substances together or separately [16 , 17 , 18 , 19]. The primary aim of the current study was to create new semen diluent formulaons for freezing ram semen collected through electroejaculaon. For this purpose, the protecve eﬀects of adding Trehalose, Iodixanol, and EDTA separately and in various combinaons to the extender were compared in the freezing of ram semen. MATERIALS AND METHODS The study received approval from the Ethics Commiee of Dicle University Health Sciences Educaon and Research Centre (2018-51333). Preparaon of extenders Semen was obtained from ﬁve Awassi rams (Ovis aries) using an electroejaculator (P-T Electronics, Model 302, Boring, OR, USA). The samples were transported to the laboratory within 30 min of collecon. Sperm concentraon was measured using an automac concentraon device (IMV, Accucell). High- quality ejaculates (concentraon: ≥ 2 × 10⁹ /mL; mass molity: ≥4; molity: ≥70%) were combined [16] and then split into 9 equal porons. Each part was prepared with its own diluent. All chemicals, except iodixanol, were obtained from Sigma Chemical Co. Visipaque 320 TM (652 mg/mL of iodixanol in water, Opakim) was used as the origin of iodixanol.Tris diluent was ulized as the basic diluent in the study. The study ulized a two-step diluon method, with glycerol introduced at +5°C [20]. The basic glycerol-free diluent was separated into 9 equal porons and the following diluent groups were formed by adding trehalose (Tr), iodixanol (Io) and EDTA alone or diﬀerent combinaons of these substances: • Tris (control), • Io5 (%5 Io, v/v) • Io10 (%10 Io, v/v), • Tr50 (50 mM Tr), • EDTA (1.5 g/L), • Io5/EDTA (%5 Io + 1.5 g/L EDTA), • TR50/EDTA (50 mM Tr + 1.5 g/L EDTA), • TR25/Io5/EDTA (25 mM Tr + %5 Io + 1.5 g/L EDTA) • TR50/Io5/EDTA (50 mM Tr + %5 Io + 1.5 g/L EDTA) Spermatological examinaons Sperm molity was evaluated aﬅer ﬁrst diluon, second diluon at +5°C and equilibraon using a phase-contrast microscope (X 400; OlympusNBX51) with a warm stage at 37°C. Molity esmates were made in three diﬀerent microscopic ﬁelds for each sample, and the esmates were averaged for the ﬁnal molity rate. Aﬅer equilibraon, straws were frozen in nitrogen vapour (4.0 cm above the liquid nitrogen) for 10 min and placed in liquid nitrogen unl evaluaon [20]. Sperm moon parameters were evaluated using a computer- assisted sperm analyser (CASA (TM) ) system (IVOS 12.3, Hamilton Thorne Biosciences) with the set-up values of Demir et al [21]. Each group had three frozen straws thawed at 37°C for 30 s in a water bath and then combined. A 10 μL sample was placed in a pre-heated 10-μm-deep Makler counng chamber (Seﬁ Medical Instruments), where sperm molity and moon parameters were analyzed at 37°C. About 600–800 sperm cells were examined across ten ﬁelds. Each CASA assessment was performed three mes per sample. Following the inial CASA analysis conducted immediately aﬅer thawing (0 h), the same semen samples were incubated at 37°C for 4 h to assess longevity. Sperm moon parameters were re-evaluated aﬅer 1, 2, and 4 h of post-thaw incubaon. Thermal stress test (TST) Aﬅer thawing, semen samples were incubated at 46°C for 15 min [16] and immediately analyzed using CASA to assess molity and moon parameters. 2 of 6

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Hypo-osmoc swelling test (HOST) The funconal integrity of the sperm plasma membrane was assessed using the modiﬁed hypo-osmoc swelling test. In summary, 20 μL of thawed semen was mixed with 350 μL of a sodium sulfate–fructose soluon (75 mOsm), incubated in a water bath at 41°C for 45 min, and ﬁxed with 50 μL of formalin- buﬀered saline. A total of 200 cells were examined under a phase-contrast microscope (X 400; Olympus, BX51) [16]. Morphological examinaons Abnormalies in the acrosome, head, mid-piece, and tail were evaluated using an eosin–nigrosin stain mixture. A total of 200 spermatozoa were examined under a light microscope (X 1000). Stascal analysis The study was conducted ten mes. Results were presented as mean ± SEM. Data were analyzed using one-way analysis of variance (ANOVA), followed by Duncan’s test to idenfy diﬀerences between groups, ulizing SPSS version 11.0 for Windows (SPSS Inc.). Diﬀerences with P-values <0.05 were considered stascally signiﬁcant. RESULTS AND DISCUSSION The results of the study was presented in 6 tables. Molity values aﬅer ﬁrs diluon, aﬅer cooling to 5 degrees and aﬅer equilibraon are presented in TABLE I. TABLE I. Comparison of molity rates in fresh semen aﬅer inial diluon, cooling to +5 °C and equilibraon between groups No Group Name Molity aﬅer ﬁrst diluaon(%) Molity at +5°C (%) Molity aﬅer equilibraon (%) 1 Control 78.8±1.11 ab 75.2±1.09 cd 72.3±2.11 cde 2 Io5 82.3±1.35 b 78.3±1.42 d 75.1±1.68 e 3 Io10 82.1±1.16 b 79.3±1.30 d 74.5±1.67 e 4 TR50 78.9±1.38 ab 70.8±2.61 bc 67.5±1.31 bcd 5 EDTA 81.5±1.05 b 78.6±1.27 d 73.3±1.83 de 6 Io5/EDTA 83.5±1.20 b 80.4±1.14 d 74.7±2.04 e 7 TR50/EDTA 79.6±1.54 ab 67.9±2.66 ab 63.5±2.48 ab 8 TR25/Io5/EDTA 79.8±1.46 ab 70.7±2.47 bc 67.2±1.12 bc 9 TR50/Io5/EDTA 75.8±2.27 a 64.2±2.97 a 58.3±2.85 a P Value P<0.05 P<0.0001 P<0.0001 Values are expressed as Mean±Standard Error (Mean±SE). abcd: Diﬀerences between values with diﬀerent leers in the same column are stascally signiﬁcant Molity of the groups containing 50 mM Trehalose aﬅer + 5°C cooling and equilibraon was lower compared the control (tris) and other study groups (P<0.0001). This may be due to the increase in osmoc pressure of trehalose, which may adversely aﬀect spermatozoa. The decrease in our results obtained from the Tr 50 group aﬅer equlibraon is similar to the results of Cirit et al [16] and Özmen et al [17]. However, while the total molity rate in the Tr 50 group aﬅer equlibraon was 80% in Cirit et al [16], this rate was found to be 67%. This may be due to the fact that the rams used in these studies were of diﬀerent breeds (Dorset, Awassi). The results of the Hypoosmoc Swelling Test (HOST) performed aﬅer thawing are presented in TABLE II. Aﬅer thawing, the Hypo-osmoc Swelling Test (HOST) results were found to be signiﬁcantly higher in all study groups (P<0.0001) compared to the control group. This shows that the substances used in the study have a posive eﬀect on membrane funcons. In studies conducted with sperm extenders containing diﬀerent doses of iodixanol and trehalose, it was determined that the control group value in the HOST tests performed aﬅer thawing was lower than the other study groups, similar to our study [15 , 16]. The results of the morphological disorder tests performed post thawing are presented in TABLE III. TABLE II. Comparison of Hypoosmoc Swelling Test (HOST) results between groups aﬅer thawing No Group İsmi HOST Posive (%) 1 Control 42.0±2.22 a 2 Io5 63.3±3.62 b 3 Io10 60.1±1.96 b 4 TR50 63.6±1.60 b 5 EDTA 63.6±2.78 b 6 Io5/EDTA 67.2±1.64 b 7 TR50/EDTA 66.5±2.99 b 8 TR25/Io5/EDTA 64.6±3.88 b 9 TR50/Io5/EDTA 61.6±1.98 b P Value P<0.0001 Values are expressed as Mean±Standard Error (Mean±SE). ab: Diﬀerences between values with diﬀerent leers in the same column are stascally signiﬁcant 3 of 6

Eﬀects of Trehalose İodixanol and EDTA on Ram Semen / Özmen et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico TABLE III. Comparison of post-thawing morphological abnormality rates between groups No Group Name Acrosome (%) Head (%) Midpiece (%) Tail (%) Total (%) 1 Control 22.5±2.37 b 1.9±0.40 b 1.6±0.30 a 2.3±0.45 a 28.3±2.52 b 2 Io5 14.8±1.03 a 0.5±0.23 a 1.3±0.43 a 1.4±0.45 a 17.9±1.21 a 3 Io10 14.8±1.51 a 0.8±0.28 a 1.3±0.30 a 1.4±0.31 a 18.2±1.48 a 4 TR50 11.3±2.20 a 0.8±1.22 a 1.5±0.44 a 2.1±0.29 a 15.6±2.23 a 5 EDTA 13.9±0.85 a 0.3±0.14 a 0.7±0.22 a 1.7±0.31 a 16.6±0.80 a 6 Io5/EDTA 13.8±1.33 a 0.3±0.19 a 0.8±0.34 a 1.3±0.33 a 16.3±1.07 a 7 TR50/EDTA 11.1±1.14 a 0.6±0.29 a 1.1±0.34 a 1.6±0.29 a 14.3±1.23 a 8 TR25/Io5/EDTA 12.4±1.22 a 0.9±0.40 a 0.8±0.18 a 1.7±0.40 a 15.8±1.24 a 9 TR50/Io5/EDTA 12.3±0.54 a 1.0±0.43 a 1.0±0.30 a 1.5±0.62 a 15.8±0.86 a P Value P<0.0001 P<0.05 P>0.05 P>0.05 P<0.0001 Values are expressed as Mean±Standard Error (Mean±SE). ab: Diﬀerences between values with diﬀerent leers in the same column are stascally signiﬁcant When the post-thaw acrosomal, head and total abnormality rates were compared, it was found that they were signiﬁcantly lower in all study groups compared to the control group (P<0.0001). There was no stascal diﬀerence between the groups in terms of midpiece and tail abnormalies (P>0.05). Considering these data, it can be said that the substances used provided protecon in general. Cirit et al [16] obtained the lowest total abnormality rates in ram sperm aﬅer thawing from 2.5% iodixanol, 5% iodixanol and 50 mm trehalose groups. The diﬀerence between these groups and the control group was found to be signiﬁcant. Swami et al [22] reported in their studies on murrah buﬀalo that 1.25%; 2.5%; 5% iodixanol did not create any diﬀerence with the control group aﬅer thawing. In our study, diﬀerences were found between all groups and the control group. These results indicate that the eﬀect of iodixanol on the morphology of spermatozoa may be species-speciﬁc. The results of the Thermal Stress Test (TST) performed aﬅer thawing were presented in TABLE IV. TABLE IV. Comparison of Thermal Stress Test (TST) results between groups aﬅer thawing No Group Name Total Molity (%) Progressive Molity (%) 1 Control 43.2±3.98 bc 16.1±2.15 ab 2 Io5 47.0±5.05 c 21.7±2.24 b 3 Io10 35.8±3.32 ab 15.8±2.14 ab 4 TR50 36.8±2.02 ab 16.5±1.21 ab 5 EDTA 36.1±1.79 ab 17.3±1.41 ab 6 Io5/EDTA 39.8±2.69 abc 22.1±1.83 b 7 TR50/EDTA 36.3±3.48 ab 14.9±2.11 a 8 TR25/Io5/EDTA 32.4±2.14 a 14.1±1.54 a 9 TR50/Io5/EDTA 30.8±4.25 a 12.8±3.02 a P Value P<0.05 P<0.05 Values are expressed as Mean±Standard Error (Mean±SE). abc: Diﬀerences between values with diﬀerent leers in the same column were stascally signiﬁcant. The highest total molity values in the post-thawing Thermal Stress Test (TST) were obtained in the Io5 group and the diﬀerence between this group and all other groups except the control and Io5/EDTA groups was signiﬁcant (P<0.05). Similarly, the highest progressive molity values were obtained from the Io5/EDTA and Io5 groups. There was a stascal diﬀerence between these groups and the TR50/Io5/EDTA, TR25/Io5/EDTA and TR50/EDTA groups, where the lowest values were obtained (P<0.05). Özmen et al.[17] found that the group containing 5% iodixanol had the best molity value aﬅer TST. When TABLE IV is examined, the best molity values aﬅer TST were obtained from the Io5 and Io5/Edta groups. These results also show that there is no negave interacon between iodixanol and edta. Total and progressive molity values aﬅer thawing (0 h) are presented in TABLE V. TABLE V. Comparison of total and progressive molity of spermatozoa at 0 h aﬅer thawing between groups No Group Name Total Molity (%) Progressive Molite (%) 1 Control 42.0±1,90 ab 22.0±1.13 cd 2 Io5 41.8±2.65 ab 20.3±1.67 bc 3 Io10 44.2±3.21 b 18.5±1.82 bc 4 TR50 37.1±2.08 ab 17.0±1.69 abc 5 EDTA 35.9±2.74 a 17.2±2.13 abc 6 Io5/EDTA 51.9±1.82 c 25.7±1.78 d 7 TR50/EDTA 39.5±2.48 ab 16.2±1.72 ab 8 TR25/Io5/EDTA 41.4±2.06 ab 16.0±1.17 ab 9 TR50/Io5/EDTA 36.3±3.41 ab 12.1±2.15 a P Value P<0.01 P<0.001 Values are expressed as Mean±Standard Error (Mean±SE). abcd: Diﬀerences between valu- es with diﬀerent leers in the same column are stascally signiﬁcant The highest total molity was obtained from the Io5/EDTA group aﬅer thawing (at the 0 h) and the diﬀerence between this group and all other groups was signiﬁcant (P<0.01). The Io5/EDTA group had the highest progressive molity value aﬅer thawing and the diﬀerence between this group and all other groups except the control group was signiﬁcant (P<0.001). These results are the most signiﬁcant ﬁndings of presented study. When added to the extender individually, iodixanol and EDTA did not have a posive eﬀect on molity aﬅer thawing. However, when used together, they signiﬁcantly increased total molity aﬅer thawing. In the study, no posive eﬀect of 5 or 10% iodixanol addion was found on total and progressive molity aﬅer thawing compared to tris diluent. This was an unexpected result because Cirit et al. [16] reported that the addion of 5% 4 of 6

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico iodixanol signiﬁcantly increased total and progressive molity aﬅer thawing in rams. This discrepancy between the results may be due to the diﬀerent iodixanol sources used. While OpPrepTM (Axis-Shield PoC AS, Oslo, Norway) was used in the study by Cirit et al. [16], Visipaque 320TM (Opakim Tıbbi Ürünler Tic. Ltd. Ş., Turkey) was used in the presented study. When these 2 iodixanol sources are compared, it is seen that some of their chemical properes diﬀerent. For example, OpPrepTM and Visipaque 320TM densies are 1.320 ± 0.001 g/ml, 320 mg I/ml; osmolality 170 ± 15 mOsm, 290 mOsm respecvely. This diﬀerences may have aﬀected the results. Within the scope of the study, total and progressive molity rates of spermatozoa were determined in the study groups at 1, 2 and 4 hours aﬅer thawing and the results were presented in TABLE VI. TABLE VI. Comparison of total and progressive molity of spermatozoa between groups at 1, 2 and 4 hours aﬅer thawing. 1st hour 1h 2st hour 2h 4st hour 4h No Group Name Total Molity (%) Progressive Molite (%) Total Molity (%) Progressive Molity (%) Total Molity (%) Progressive Molity (%) 1 Control 21.5 ±3.25 a 12.2±1.99 19.6±2.49 5.3±0.75 7.2±0.84 a 1.4±0.28 ab 2 Io5 26.0±2.66 ab 12.1±1.58 25.4±4.90 8.1±1.46 17.5±4.42 bc 2.7±0.63 abcd 3 Io10 32.3±3.72 abc 13.8±1.64 23.8±3.62 6.9±1.31 12.1±2.91 abc 1.7±0.37 abc 4 TR50 28.8±3.68 abc 10.8±2.46 27.4±4.41 5.0±0.82 8.5±1.60 ab 1.2±0.25 a 5 EDTA 33.5±4.58 bc 16.0±2.59 22.1±3.48 10.1±2.30 16.4±3.16 abc 5.6±1.74 d 6 Io5/EDTA 34.1±4.08 bc 15.3±2.42 20.0±4.00 8.8±2.05 9.5±1.73 ab 3.4±0.98 abcd 7 TR50/EDTA 38.8±4.05 c 13.9±2.71 22.7±4.76 9.0±2.08 15.1±2.96 abc 4.3±1.09 bcd 8 TR25/Io5/EDTA 35.8±2.88 bc 14.7±2.24 27.3±5.22 11.2±2.89 17.6±3.63 bc 4.4±1.18 bcd 9 TR50/Io5/EDTA 30.2±4.15 abc 13.4±2.87 24.8±4.83 8.9±2.07 20.1±3.65 c 4.7±1.15 cd P Value P<0.05 P>0.05 P>0.05 P>0.05 P<0.05 P<0.01 Values are expressed as Mean±Standard Error (Mean±SE). abcd: Diﬀerences between values with diﬀerent leers in the same column are stascally signiﬁcant The highest total molity values at the end of the 4 h were obtained from TR50/Io5/EDTA, TR25/Io5/EDTA and Io5 groups (20.1, 17.6 and 17.5%, respecvely) and the diﬀerences between these groups and the control group (7.2%) were stascally signiﬁcant (P<0.05). In the resilience test, EDTA and TR50/Io5/ EDTA groups had the highest progressive molity values at the end of the 4 h (5.6 and 4.7%, respecvely) and the diﬀerences between these groups and control (1.4%) and TR50 groups (1.2%) were found to be signiﬁcant (P<0.05). According to the data of Aisen et al. [12], the TR50/EDTA group, which was expected to be one of the best, did not produce the claimed synergisc eﬀect. However, the Io5/EDTA group had higher values for molity aﬅer equilibraon, aﬅer TST and aﬅer thawing than the TR50/EDTA group (P<0.01). These data indicate that there is a synergisc interacon between EDTA and iodixanol and that this posive eﬀect is more pronounced than the Trehalose/EDTA combinaon. However, there are also diﬀerences between the studies, such as the method of sperm collected (arﬁcial vagina, electroejaculaon) and the diﬀerent breeds of rams (Pampinta, Awessi). In addion, they show a path for further research in this area. 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