Special Issue:

Advancements in Animal Health and Production in Low and Middle-Income Countries

Quality of Tomcat Sperm Recovered by Castration after Using the FertiCult Flushing Medium

Asmaa S. Al-Alywi, Abeer A. Yaseen, Nagham Y. Abood, Mohammed R. Abduljaleel*

Department of Surgery and Obstetrics, College of Veterinary Medicine, University of Basrah, Basrah, Iraq.

Received | June 24, 2025; Accepted | August 04, 2025; Published | August 16, 2025

*Correspondence | Mohammed R. Abduljaleel, Department of Surgery and Obstetrics, College of Veterinary Medicine, University of Basrah, Basrah, Iraq; Email: [email protected]

Citation | Al-Alywi, A.S., Yaseen AA, Abood NY, Abduljaleel MR (2025). Quality of tomcat sperm recovered by castration after using the ferticult flushing medium. J. Anim. Health Prod. 13(s1): 173-180.

DOI | https://dx.doi.org/10.17582/journal.jahp/2024/13.s1.173.180

ISSN (Online) | 2308-2801

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Semen collection techniques fall into two categories including in vitro collection (testicular or primary epididymal semen) and in vivo collection (mostly ejaculated semen). In 2003, Tsutsui et al. have highlighted that the tomcat (Felix catus) may produce high-quality ejaculates in vivo by copulation using an artificial vagina, urethral catheterization, or electroejaculation. Given that there are many testes accessible following routine castration surgeries (Zambelli and Cunto, 2006; Pisu et al., 2017), the use of epididymal spermatozoa (ES) could be advantageous when needed for research purposes on assisted reproductive technologies (ARTs) (sperm freezing, artificial insemination, in vitro embryo production). In addition, the collection of ES is of particular importance in the event of the sudden death of valuable individuals and, more often, for the conservation of gametes of endangered species (Brunn, 2022). Epididymal mincing, in vitro epididymal sperm aspiration post-epididymectomy, and in vivo percutaneous epididymal sperm aspiration (PESA) are some of the techniques used to collect epididymal semen. After an orchiectomy or post-mortem, epididymal mincing, also known as the float-up procedure, is a reliable and non-repetitive method for collecting specimens (Tebet et al., 2006). After being held in a liquid (usually a semen extender), the epididymis is separated from the testicle and cut in parallel. The sperm cells can be discharged into the media once the epididymis is incubated for a few minutes (Simons et al., 2019; Mohsen et al., 2024). Tomcats reach sexual maturity between 7 and 12 months of age, although spermatogenesis takes approximately 46.8 days and can begin as early as 5 months of age Siemieniuch and Woclawek-Potocka (2007); França and Godinho (2003). According to Tsutsui et al. (2004), over 80% of cats exhibit complete spermatogenesis and epididymal sperm stores beginning at 7 months. The benefit of cryopreserving semen is that it allows genetic material to be transferred between distant places, not just between living animals, ensuring that genetic variety is preserved even if the animals pass away before reproducing (Vansandt et al., 2021).

When animals or sperm donors passed away unexpectedly or were castrated for medical reasons, epididymal sperm were collected from the caudal or corpus areas. Kunkiti et al. (2016) and Chatdarong et al. (2016) reported suitable substitutes to preserve the priceless genetic components. Tsutsui et al. (2003) reported a successful pregnancy in domestic cats following the use of cryopreserved sperm cells from the caudal epididymides for unilateral intrauterine artificial insemination. Conversely, 1-(5-oxohexyl)- 3,7-dimethylxanthine is the chemical name for Pentoxifylline, a dimethylxanthine derivative. By blocking cAMP phosphodiesterase activity, the PX stops cAMP breakdown and, it is assumed, promotes sperm motility (Steiber et al., 2004). Furthermore, because PX scavenges ROS and subsequently lowers lipid peroxidation, it protects sperm membranes (i.e., maintaining the functional membrane integrity of the sperm tail) (Seim et al., 2002).

Aims of this study were to highlight the benefits of sperm cells collection from castration surgery and to establish a protocol for semen cooling tom cat., Additionally, we aim to investigate the cooling ability of semen tom cat on specific physical, parameters (sperm motility and number) and a comparative study of Pentoxifylline and Flushing medium effects on semen quality tom cat before and after cooling process different time.

MATERIALS and METHODS

Animals

According to the University of Basrah’s College of Veterinary Medicine’s BCVM standards, the study was approved by an ethics committee with a number (81/37) in 2025. For this investigation, twenty sexually mature tomcats (Felis catus) were chosen based on their reproductive and clinical evaluations. They were 12 to 18 months old on average. A comprehensive clinical examination and a macroscopic evaluation of the reproductive organs of the research animals were performed during the preoperative phase, including palpation and inspection. We only employed clinically healthy animals that had normal testicular symmetry and consistency and showed no damage to their external reproductive organs. The study’s approved cats underwent bilateral Feline castration (Orchiectomy) surgery after a 12-hour fast from food and a 6-hour fast from water (Woodruff et al., 2015). Antibiotic therapy with penicillin (IM), meloxicam (0.2 mg/kg IM) (Alrafas et al., 2023; Mohsin et al., 2025), 13 mg of ketamine, and 5 mg of xylazine comprised the preoperative protocol used in the study (Jassim et al., 2023). General anaesthesia was administered within the desired time frame, following a bilateral orchiectomy (Miller et al., 2018; Abduljaleel, 2024). The testis-epididymis complexes (TEC) of the animals’ left and right were acquired. After recuperating from the anaesthetic, the animals were released from the College, and post-surgical therapy instructions were given to the owners. Following collection, the proximal area of the deferent duct and the epididymal cauda were used to retrieve the epididymal sperm using the slicing procedure (Figure 1). A knife was used to cut off the contours of the epididymal duct after the epididymis’s surrounding connective tissue was removed (Smeak and Hazenfield, 2015). The outside of the epididymis was then cleaned with a saline solution heated to 37°C. Each complete epididymal duct was put in a petri plate that had also been heated to 37°C after the epididymis had been cleaned externally. The proximal section of the deferent duct and the epididymal cauda were ultimately sliced into tiny pieces with two scalpel blades and left in the Petri dish for a minute in order to encourage contact between the sperm

 

and the extender. Sperm were evaluated using aliquots of the resultant diluent (semen samples were obtained from castration-tom cats and diluted with Tris-citric egg yolk-glycerol) and diluent treated with either flushing medium or Pentoxifylline (PX). In a phase contrast microscope, 10 μL of the dilution was examined using a computer system analysis (CASA) video camera. The spermatic parameters listed below were analyzed: Total and progressive motility, average path velocity( VAP), curvilinear velocity (VCL), straight line velocity (VSL), linearity index (LIN) and straightness index (STR), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) (Mota Filho et al., 2014). A 10µl aliquot of the (Tris-citric egg yolk-glycerol) diluter and diluent, supplemented with Pentoxifylline ( PX) or flushing medium, was used to assess the total motility of the epididymal sperm.

Preparation of pentoxifylline (PX).

10 mg of PX powder (Sanofi Aventis, Egypt) was dissolved in 10 ml of PBS (0.1%) and stirred until a homogeneous solution was formed. Every day, under sterile conditions, these concentrations were prepared using a Millipore filter (0.45 μm) and UV light (Al-Dujaily et al., 2012).

Flushing medium (FertiCultTM contains phenol red)

HEPES, buffered medium, physiologic salts, glucose, lactate, pyruvate, bicarbonate, and human serum albumin (4.0g/l), a medicinal substance derived from human blood plasma, are all present in FertiCultTM Flushing media (Figure 2).

 

Extender preparation

In 100 milliliters of distilled water, 3.028 grams of trishydroxymethyl-aminomethane, 1.78 grams of monohydrated citric acid, and 1.25 grams of fructose were dissolved to create the stock solution with a 6.6 pH (Vizuete et al., 2014). The three final doses of glycerol (4, 6, and 8%) were then used to create experimental treatments after 20% of the fluid was replaced with egg yolk. Tylosin (100 μg/mL), Gentamicin (500 μg/mL), and Lincomycin (300/600 μg/mL) were added as antibacterial.

Evaluation of semen quality

Physical properties

Sperm concentration: Computer-assisted semen analysis (CASA) was used to measure the sperm concentration in ejaculates (Baracaldo et al., 2007). Assays for motility: A computer-assisted sperm analysis (CASA) system was used to assess motion characteristics, which had been previously validated in our laboratory (Gadea et al., 2005). Amplitude of lateral head displacement (ALH, μm), wobble of the curvilinear trajectory (WOB, ratio of VAP/VCL%), curvilinear velocity (VCL, μm/s), straight-line velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity of the curvilinear trajectory (LIN, ratio of VSL/VCL%), straightness (STR, percentage of VSL/VAP%), and the percentage of motile spermatozoa (%motil) were the motility characteristics derived from CASA. Prior to (just ejaculated) and following sperm therapy, a 5μl drop of the sample was put on a heated (37 °C) slide and covered with a cover slip. To count spermatozoa, specific conditions must be met (Axnér et al., 2004). The percentage of dead sperm was calculated using the methodology of Gervasi and Visconti (2017). After the smears were dried, an examination was conducted under a light microscope at 400X. To accomplish this, 1-2 drops of the freshly drawn semen sample and 1-2 drops of eosin-nigrosine that had been warmed up were placed on a sterile slide. The semen sample and stain were then combined on the edge of the second slide, and the mixture was then dragged along the surface of the clean slide. In order to make the dead sperms turn red while the living sperms do not, nigrosine is used to stain the background and eosin to stain the dead sperms.

The stain was prepared as follows

The active ingredient of the stain Eosin (1.67 gm), Nigrosine (2-10 gm) and Sodium citrate (2.9-3 gm) were dissolved in 100 millilitres of double-distilled water, followed by heating, filtering, and combining. A heated stage was included in the study’s microscope, and the Eosin-Nigrosine stain was pre-warmed at 37 °C in a water bath.

Experimental design: Using a plastic bag, semen samples were taken from castrated Tom cats. The samples were then physically examined and diluted using a Tris-citric egg yolk-glycerol diluter. Pentoxifylline (PX) or flushing medium were added to the same diluent at varying amounts. The following procedures were used to prepare twenty tom cats for chilling after they were removed from the study animals. A physical evaluation of the ejaculate was conducted, including sperm concentration, progressive motility percentage and dead sperm percentage.

• Dilution: The concentration of sperm in the ejaculates was adjusted using Tris diluent. The diluter and ejaculates were then placed in a water bath at 37°C. The diluted semen sample was divided into two equal portions using a plastic test tube to add antioxidants. One was included with Pentoxifylline (5 mmol/mL) and 5 mL of diluent (T1 Tris) while the other one was added Flushing medium (including Phenol Red from FertiCultTM). For four hours, diluted semen with varying antioxidant concentrations was kept at the same temperature (5 °C).
• Forty epididymides were gathered and the following comparison was made between two experimental groups: Tris+pentoxyfylline (T1) was used to recover the sperm from twenty epididymides that had been chilled to 5°C at 48, 72, 96, and 120 hours; FertiCult TM Flushing medium (T2) was used to recover the sperm from twenty epididymides that had been cooled to 5°C at 0, 48, 72, 96, and 120 hours. Fresh sperm from ten epididymides were recovered using 0 hours (control contains only Tris-citric egg yolk-glycerol (TCEG). Each extension and cooling time had a total of 40 epididymides.
• Statistical analysis: SPSS (Statistical Package for the Social Sciences, Version 17, Illinois, USA) was used to collect and analyze raw data for descriptive statistics, including means and standard error of the Mean (SE). A P value of less than 0.05 was deemed statistically significant (Jasim et al., 2025).

RESULTS and DISCUSSION

In the pentoxifylline experiment, recovery of epididymal sperm from in-tom cats revealed a greater non-significant mean total motility between (0 and 48;96 and 120 hr) (76.29±0.24 and 72.04±0.21); (59.78±0.31 and 56.92±0.18).In comparison to cooling at 120 hours (92.45±0.21; 32.81±0.65), the mean sperm concentration and VSL (µm/s) did not significantly change between 0, 48, 72, and 96 hours (137.94±0.31, 126.83±0.27, 109.58±0.38; 49.92±0.41, 47.18±0.25, 40.98±0.34, and 110.26±0.56; 40.3855±0.56). Fresh progressive motility and 48 hours were shown to be higher than the cool times (72, 96, and 120 hours) by computer analysis (52.07±0.66, 46.2255±0.66, and 43.15±0.42). The VAP (µm/s) of the Tris extender was significantly higher at 0 and 48 hours (46.15±0.42 and 46.03±0.36) than it was at 120 hours (40.09±0.47). However, a significant decrease in fresh recovery sperm following cooling for VCL with Tris with Pentoxifylline was observed at 96 and 120 hours (113.4130±0.07 and 119.98±0.52) as opposed to 60 and 72 hours (96.95±0.38 and102.09±0.34). Regarding Tris and Tris with Pentoxifylline, LIN(%) was lower at 0 hours (51.37±0.53) than cooling at 48 and 72 hours (44.72±0.42 and 39.30±0.54°C), and there was no significant difference between 96 and 120 hours (30.7775±0.43 and 27.68±0.90). Table 1 shows the comparison between Tris and Tris with Pentoxifylline, while the other parameters (AIH (µm), BCF(HZ), and STR(%)did not change with the various cooling durations for each extender. The study’s findings on the percentage of dead sperm at various cooling times during sperm processing are presented in Tables 1 and 2.

At 0 hours, the percentage of dead sperm was at its lowest (14.21 ± 0.098%), and it was substantially different from that at 120 hours (P ˂ 0.05). Meanwhile, upon cooling for 96 and 120 hours, the results were 31.71 ± 0.28 and 34.11 ± 0.27%, respectively. The maximum number of dead sperm was recorded at 96 and 120 hours(31.71 ± 0.28 % and

 

Table 1: Effect of Pentoxifylline diluted or Tris on the tomcat epididymal sperm parameters determined after cooling of epididymides assessed by CASA. for Mean±S.E.

Cooling time				0 hour (control contains only (Tris-citric egg yolk-glycerol (TCEG)	Parameter pentoxifylline
120 hour	96 hour	72 hour	48 hour
34.11±0.27A	A0.28±31.71	24.05±0.08B	18.16±0.092C	14.21±0.098C	Dead sperm %
56.92±0.18C	59.78±0.31C	66.73±0.23B	72.04±0.21A	76.29±0.24A	Total motility%
92.45±0.21C	110.26±0.56B	109.58±0.38B	126.83±0.27A	137.94±0.31A	Sperm concentration
43.15±0. 42C	46.2255±0.26BC	52.07±0.66B	57.88±0.20A	61.83±0.61A	Progressive motility %
40.09±0.47C	42.3285±0.33BC	44.85±0.33B	46.03±0.36A	46.15±0.42A	VAP (µm/s)
32.81±0.65C	40.3855±0.56B	40.98±0.34B	47.18±0.25A	49.92±0.41A	VSL (µm/s)
119.98±0.52A	113.4130±0.07A	102.09±0.34B	88.54±0.47C	96.95±0. 38B	V CL (µm/s)
3.12±0.12A	3.1620±0.09A	2.38±0.06B	2.47±0.082B	1.25±0.05C	AIH (µm)
14.25±0.07A	12.4090±0.06A	10.09±0.01A	3.43±0.073B	2.22±0.06B	BCF (HZ)
27.68±0.90D	30.7775±0.43D	39.30±0.54C	44.72±0.42B	51.37±0.53A	LIN(%)
82.47±0.53B	85.2550±1.02AB	88.28±0.65A	89.34±0.67A	91.80±0.70A	STR(%)

 

The amplitude of lateral head displacement (ALH), beat cross frequency (BCF), linearity index (LIN), straightness index (STR), average path velocity (VAP), curvilinear velocity (VCL), and straight line velocity (VSL). Statistically significant variations between extenders for the various cooling durations are indicated by different capital letters within a row (P < 0.05).

 

Table 2: Effect of ferticult flushing medium or Tris on the tomcat epididymal sperm parameters determined after cooling of epididymides assessed by CASA for Mean±S.E.

Cooling time				0 hour(control contains only (Tris-citric egg yolk-glycerol (TCEG)	Parameter Ferticult flushing medium
120 hour	96 hour	72 hour	48hour
15.69±0.071A	13.05±0.041A	10.18±0.053B	9.15±0.063B	7.97±0. 049C	Dead sperm %
75.63±0.29C	77.28±0.21B	80.29±0.37A	81.47±0.18A	82.09±0.24A	Total motility%
180.02±0.45C	186.91±0.24C	191.58±0.43C	207.72±0.19B	235.36± 0.26A	Sperm concentration
64.12±0.28B	66.83±0.42B	69.53±0.38A	69.97±0.30A	71.47±0.53A	Progressive motility %
38.84±0.45BC	40.60±0.60B	42.30±0.07B	46.24±0.29A	47.94±0.37A	VAP(µm/s)
31.26±0.56B	39.91±0.49A	39.10±0.63A	41.10±0.28A	45.92±0.29A	VSL(µm/s)
138.97±0.31A	138.80±0.47A	136.29±0.34A	128.75±0.46B	131.02±0.56B	V CL(µm/s)
2.55±0.07A	3.60±0.08A	3.33±0.07A	2.67±0.05B	1.94±0.09C	AIH(µm)
17.30±0.07A	15.37±0.08A	13.39±0.06A	7.45±0.07B	3.25±0.06C	BCF(HZ)
21.85±1.03C	29.27±0.42B	30.11± 0.53AB	32.56±0.67A	34.29±0.07A	LIN(%)
83.61±0.73B	91.24±0.54A	93.57±0.53A	93.99±0.49A	94.08±0.51A	STR(%)

 

The amplitude of lateral head displacement (ALH), beat cross frequency (BCF), linearity index (LIN), straightness index (STR), average path velocity (VAP), curvilinear velocity (VCL), and straight line velocity (VSL). Statistically significant variations between extenders for the various cooling durations are indicated by different capital letters within a row (P < 0.05).

 

34.11±0.27%), which was substantially different from the 0-hour value in the Pentoxifylline experimental group. Pentoxifylline (PX) has been shown to enhance testicular sperm motility (Sato and Ishikawa, 2004), as well as hyperactivation and the acrosome reaction after activation (Abid, 2005; Al-Dujaily et al., 2007). When spermatozoa were washed and activated in vitro using FertiCult medium TM, active sperm motility was observed. This may be related to the ingredients in FertiCult flushing medium, which comprise a mixture of human serum albumin (4.00 g/L), glucose, lactate, physiologic salts, bicarbonate, and HEPES (Dickey et al., 2009). As a buffering system, HEPES, when present in the media alone, enhanced the sperm’s buffering ability. The statistics in this study, however, indicate that the cooling process causes the percentage of dead sperm to rise. “PTX can be used as a potent antioxidant substance against oxidative stress and subsequent effects to reduce abnormal sperm and dead count in tom cats,” according to Cooper (2010). The longer the storage period, the higher the abnormality rate, which is attributed to cold shock and an imbalance in osmotic pressure resulting from metabolic processes during storage at 5°C, according to Yani and Nuryadi (2001). The results showed that the lowest significant value (p < 0.05) occurred at 0 hours, while the greatest non-significant value (p < 0.05) occurred at 96 and 120 hours (13.05 ± 0.041% and 15.69 ± 0.071%, respectively) in the ferticult flushing medium experiment. The results showed a significant (P < 0.05) increase in dead sperm. Table 2 and Figure 3 show the percentage of tomcat spermatozoa after cooling at 120 hours (15.69±0.071%) compared to after cooling at 0 hours (7.97±0.049%). When a high genetic value male is castrated (electively or in an emergency), the epididymis includes a valuable source of sperm that may be recovered. As sperm from a testis can only be obtained once, the most effective method for maximizing the recovery, preservation (germplasm banking), and utilization of gametes through assisted reproductive technologies must be employed. In small and laboratory animals, the only method for collecting epididymal spermatozoa is through the ductus deferens, which has a smaller diameter (Ali et al., 2021). Our study examined the effects of cool storage after 48, 72, 96, and 120 hours of storage. The initial evaluation was conducted at day 5 (120 hours) using a difficult-to-flush medium; however, our findings suggest that motility may not have decreased even before 72 hours of incubation. Previous research has examined the impact of cool storage on the quality of tomcat sperm (Angrimani et al., 2018).

 

The results of our study also showed that the epididymal sperm from the tomcat in the experiment ferticult-flushing medium analyzed showed no significant differences (P >0.05) regarding four of the parameters (motility, progressive motility, VSL (µm/s), and STR (%). It also showed a non-significant difference between 0 hr (81.09±0.24, 71.47±0.53, 45.92±0.29, and 94.08±0.51), 48 hr (81.47±0.18, 69.97±0.30, 41.10±0.28, and 93.99±0.49), and 72 hr (80.29±0.37, 69.53±0.38, 39.10±0.63, and 93.57±0.53) of cooling time. The assessed groups listed in Table 2 did not differ in these values (P > 0.05), including Mean (± S.E.) sperm concentration, progressive motility, cooling sample VCL (µm/s), AIH (µm), and BCF (Hz). The values of sperm parameters after varying cooling times were found to be non-significantly different between 96 (186.91±0.24; 66.83±0.42; 138.80±0.47; 3.60±0.08 and 15.37±0.08) and 120 hours (180.02±0.45; 64.12±0.28; 138.97±0.31; 2.55±0.07 and 17.30±0.07). Our study’s results verified that the epididymal sperm from the tom cat in the experiment’s ferticult-flushing medium analysis demonstrated that the chilling procedure had no discernible impact on the sperm characteristics, and the ferticult-flushing medium was the same for all cooling times. These studies show a gradual deterioration in semen quality (measured by sperm parameters). For seven days, male cat semen was stored at 5°C in cold storage for this study. The findings of this investigation align with those of earlier research. Perez-Marin et al. (2017) kept the tomcat epididymides at 4 °C for 48 hours. At the time of evaluation, the scientists found that the acrosomal integrity of epididymal spermatozoa was unaffected by the cool storage. During the 72-hour storage period, the acrosome integrity of tomcat epididymal spermatozoa was not significantly impacted by cool storage at 5°C, according to Angrimani et al. (2018). For tomcat spermatozoa, a similar study by Gañán et al. (2009) found no discernible impact of cool storage at the same temperature (5°C) over 72 hours. In contrast to earlier studies, Woodruff et al. (2015) examined the impact of cool storage on the characteristics of tomcat spermatozoa. However, the sperm samples were kept at 4°C for ten days. This study was the first to use ferticult-flushing media to recover the epididymal sperm after cooling the epididymis of domestic cats for up to 120 hours. The substantial improvement in sperm viability after the chilling time was most likely due to the components of the difficult-flushing medium, in particular. In addition, the samples obtained using the ferticult-flushing media exhibited greater motility values and progressive motility than the slices. Even though the sperm were not impacted by the various chilling times caused by the castration collection methods, similar outcomes were noted in Tom Cats, where samples obtained by castration demonstrated 82.09±0.24% higher motility and 71.47±0.53% higher viability (Turri et al., 2012). However, the samples in this investigation showed increased Mean (±S.E.) sperm concentration, Progressive motility, V CL (µm/s), AIH (µm), and BCF (Hz) in the cooling samples. In line with the findings of Simons et al. (2019), who noted higher VAP and ALH in cooling-collected samples, a comparison of sperm parameter levels after varying cooling times revealed no significant difference between 96 and 120 hours. Because the freeze/thaw process greatly reduces sperm activity, fresh spermatozoa are of higher quality than frozen samples (Clark and Swain, 2014; Terrell et al., 2012). The composition of FertiCult flushing medium includes a mixture of human serum albumin (4.00 g/L), glucose, lactate, physiologic salts, bicarbonate, and HEPES (Dickey et al., 2009). When used as a buffering system, HEPES alone in the media enhanced the buffering capacity and pH stability between 7.2 and 7.6. To prevent the pH from falling below 7.0, no CO2 incubation is necessary, as this enables the media to withstand pH variations caused by changes in cellular metabolism more effectively (Clark and Swain, 2014). According to Al-Sultani et al. (2013), the current study found that FertiCult sperm washing medium TM has a higher impact on particular male sperm function metrics than other sperm washing media.

CONCLUSIONS and RECOMMENDATIONS

The study was the first to use a ferticult-flushing medium to recover epididymal sperm and cool the epididymis of domestic cats for up to 120 hours. The significant increase in sperm viability following the chilling period was probably caused by the components of the ferticult-flushing solution, in particular. In our investigation, we found that incubation for 120 hours did not affect total motility. However, the percentage of progressive motile cells was not significantly reduced in the group incubated for 120 hours before cooling with ferticult-flushing medium when compared to cool samples with Pentoxifylline. These data indicate that when transporting the heritable material of a freshly dead sample (e.g., a castrated individual) to the laboratory. For future, we recommend making straws, freezing them, and then thawing them in a Ferticult flushing medium. We recommend studying the deformities after the cooling, freezing and thawing process of castrated tomcats. Also, we suggest the integrity of the sperm membrane using a fluorescent microscope.

ACKNOWLEDGEMENTS

We want to thank the Head of the Department of Surgery and Obstetrics, the Dean of Veterinary Medicine, and the President of Basrah University for their support of the college laboratory.

NOVELTY STATEMENT

The novelty of our work entitled (Quality of tom cat sperm recovered by castration after using the FertiCult flushing medium) lies in its investigation of a novel method for sperm recovery in tom cats. Specifically, we explore the efficacy of using FertiCult flushing medium during castration to optimize the retrieval of viable sperm. This approach presents a significant departure from traditional sperm collection methods in felines, potentially offering a more efficient and less invasive alternative for genetic resource banking and assisted reproduction in this species.

AUTHOR’S CONTRIBUTION

All authors equally contributed.

Conflict of interest

The authors have declared no conflict of interest.

References

Abduljaleel MR (2024). Xylazine-ketamine outperforms diazepam-ketamine in rabbit anesthesia. Acad. Open, 9: 1-9. https://doi.org/10.21070/acopen.9.2024.9969

Abid RS (2005). Effect of pentoxifylline (Trental®) on human sperm in vitro activation of asthenospermic infertile patients. High diploma thesis, Institute of Embryo Research on Infertility Treatment, Al- Nahrian University.

Al-Dujaily SS, Al-Nakash AR, Al-Biaty SA (2007). Effect of Pentoxifylline on the outcome of artificial inseminations. Iraqi Postgrad. Med. J., 5: 377-383.

Al-Dujaily SS, Al-Shahiri N, Abbas A (2012). Effect of L-carnitine on in vitro sperm activation of infertile men. I J. Embry. Int. Res., 2: 22-25. https://pmc.ncbi.nlm.nih.gov/articles/PMC4163266/

Ali HH, Domain G, Luvoni GC, Chaaya R, Van Soom AWE (2021). Canine and feline epididymal semen Aplentiful source of gametes. Animals, 11: 1-10. https://doi.org/10.3390/ani11102961

Alrafas HR, Alahmed JAS, Essa IM, Kadhim SZ, Al-Tameemi HM, Abduljaleel MR, Al-Hejjaj MY (2023). Role of anti-inflammatory interleukin 10 in asymptomatic heartworm infection (Dirofilariasis) in dogs. Adv. Life Sci., 10: 412-417. https://doi.org/10.62940/als.v10i3.1817

Al-Sultani YK, Al-Katib SR, Al-Zayadi S (2013). Effect of vitamin C on in-vitro sperm activation of Asthenozoospermic infertile patients. Am. J. Res. Commun., 1: 40-48. http://www.usa-journals.com/wp-content/uploads/2013/09/Al-Sultani_Vol110.pdf

Angrimani DDSR, Nagai KK, Rui BR, Bicudo LC, Losano JDDA, Brito MM, Nichi M (2018). Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours). Reprod. Domest. Anim., 53: 163-170. https://doi.org/10.1111/rda.13086

Axnér E, Hermansson U, Linde-Forsberg C (2004). The effect of Equex STM paste and sperm morphology on post-thaw survival of cat epididymal spermatozoa. Anim. Reprod. Sci., 84: 179-191. https://doi.org/10.1016/j.anireprosci.2003.11.003

Baracaldo M, Barth A, Bertrand W. (2007). Steps for Freezing Bovine Semen: From Semen Collection to the Liquid Nitrogen Tank. IVIS Reviews in Veterinary Medicine. https://www.ivis.org/library/reviews-veterinary-medicine/steps-for-freezing-bovine-semen-from-semen-collection-to-liquid

Brunn A (2022). Surgical castration in dogs: does the incision approach influence postoperative recovery? Vet. Evid., 7: 1-7. https://doi.org/10.18849/ve.v7i4.587

Chatdarong K, Thuwanut P, Morrell JM (2016). The development of cat testicular sperm cryopreservation protocols: Effects of tissue fragments or sperm cell suspension. Theriogenology, 85: 200-206. https://doi.org/10.1016/j.theriogenology.2015.09.030

Clark NA, Swain JE (2014). Buffering systems in IVF. Culture media, solutions, and systems in human ART, by P. Quinn (1st ed), Company Press, pp. 30-46. https://doi.org/10.1017/CBO9781139059053.005

Cooper TG (2010). The epididymis, cytoplasmic droplets and male fertility. Asian J. Androl., 13: 130–138. https://doi.org/10.1038/aja.2010.97

Dickey P, Brinsden PR, Pyrzak R (2009). Manual of intrauterine insemination and ovulation induction. Cambridge University Press. https://doi.org/10.1017/CBO9780511642159

Franca LR, Godinho CL (2003). Testis morphometry, seminiferous epithelium cycle length, and daily sperm production in domestic cats (Felis catus). Biol. Reprod., 68: 1554–1561. https://doi.org/10.1095/biolreprod.102.010652

Gadea J, Gumbao D, Matas C, Romar R (2005). Supplementation of the thawing media with reduced glutathione improves function and the in vitro fertilizing ability of boar spermatozoa after cryopreservation. J. Androl., 26: 749-756. https://doi.org/10.2164/jandrol.05057

Gañán, N, Gomendio M, Roldan ERS (2009). Effect of storage of domestic cat (Felis catus) epididymides at 5 C on sperm quality and cryopreservation. Theriogenology, 72, 1268-1277. https://doi.org/10.1016/j.theriogenology.2009.07.023

Gervasi MG, Visconti PE (2017). Molecular changes and signaling events occurring in spermatozoa during epididymal maturation. Andrology, 5: 204–218. https://doi.org/10.1111/andr.12320

Jasim MM, Naeem RM, Abduljaleel MR, Sanad NH, Ibrahim AA (2025). Efficacy of autogenic, allogenic and heterogenic platelet rich plasma (PRP) on Avulsion skin wounds in rabbit model. Adv. Life Sci., 12: 91-97. https://doi.org/10.62940/als.v12i1.2907

Jassim MM, Abduljaleel MR, Abdulkareem ZB, Sanad NH, Alrashid IMH (2023). Study the effect of the magnetic field on the healing of bone fracture after implant avian bone in femoral bone in rabbits. Adv. Anim. Vet. Sci., 11: 1779-1784. https://doi.org/10.17582/journal.aavs/2023/11.11.1779.1784

Kunkitti P, Bergqvist AS, Sjunnesson Y, Johannisson A, Axnér E (2016). The tolerance of feline corpus and cauda spermatozoa to cryostress. Theriogenology, 85: 502-508. https://doi.org/10.1016/j.theriogenology.2015.09.032

Miller KP, Rekers WL, DeTar LG, Blanchette JM, Milovancev M (2018). Evaluation of sutureless scrotal castration for pediatric and juvenile dogs. J. Am. Vet. Med. Assoc., 253: 1589-1593. https://doi.org/10.2460/javma.253.12.1589

Mohsen AK, Jassim SE, Alaridhi TT, Al-Erjan AM, Lahhob QR, Mudhafar M (2024). Revolutionizing infectious disease management in animals through advanced phage therapy: A comprehensive strategy for the eradication of multidrug-resistant bacterial pathogens. J. Anim. Health Prod. 12(s1): 157-167. https://doi.org/10.17582/journal.jahp/2024/12.s1.157.167

Mohsin TA, Abduljaleel MR, Radhi AJ, Abbas MF, Alrashid IMH, Khudhair ZW (2025). Histopathological effects of streptomycin treatment on macrophages in lymph nodes, spleen, liver and kidneys of rats. Adv. Anim. Vet. Sci., 13: 1337-1345. https://doi.org/10.17582/journal.aavs/2025/13.6.1337.1345

Mota-Filho AC, Silva HVR, Nunes TGP, de Souza MB, de Freitas LA, de Araújo AA, da Silva LDM (2014). Cryopreservation of canine epididymal sperm using ACP-106c and TRIS. Cryobiology, 69: 17-21. https://doi.org/10.1016/j.cryobiol.2014.04.013

Perez-Marin CC, Jimenez E, Aguera EI (2017). Effects of epididymis cold storage on frozen-thawed epididymal sperm quality in tomcats (Felis catus). Veterinární medicína, 62, 147-152. DOI: 10.17221/253/2015-VETMED

Pisu MC, Ponzio P, Rovella C, Baravalle M, Veronesi MC (2017). Usefulness of an injectable anaesthetic protocol for semen collection through urethral catheterization in domestic cats. J. Feline Med. Surg., 19: 1087–1090. https://doi.org/10.1177/1098612X16679589

Sato M, Ishikawa A (2004). Room temperature storage of mouse epididymal spermatozoa: exploration of factors affecting sperm survival. Theriogenology, 61, 1455-1469. https://doi.org/10.1016/j.theriogenology.2003.07.013

Seim H, Kiess W, Richter T (2002). Effects of oral L-carnitine supplementation on in vivo long-chain fatty acid oxidation in healthy adults. Metab. Clin. Exp., 51: 1389-1391. https://doi.org/10.1053/meta.2002.35181

Siemieniuch MJ, Woclawek-Potocka I (2007). Morphological features of the seminiferous epithelium in cat (Felis catus, L. 1758) testes. Journal of Reproduction and Development, 53, 1125-1130. https://doi.org/10.1262/jrd.19053

Simons N, Van Soom A, Domain E, Wydooghe E (2019). Collection and cryopreservation of epididymal spermatozoa in dogs. Vlaams Diergeneeskundig Tijdschrift, 88: 77-82. https://doi.org/10.21825/vdt.v88i2.16028

Smeak DD, Hazenfield KM (2015). Creating a leak-proof ligature with confidence, part 1: Overview of ligation and surgical binding knots. Todays Vet. Pract., 5: 46-49.

Steiber A, Kerner J, Hoppel CL (2004). Carnitine: A nutritional, biosynthetic, and functional perspective. Mol. Aspects Med., 25: 455-473. https://doi.org/10.1016/j.mam.2004.06.006

Tebet JM, Martins MIM, Chirinea VH, Souza FFD, Campagnol D, Lopes MD (2006). Cryopreservation effects on domestic cat epididymal versus electroejaculated spermatozoa. Theriogenology, 66: 1629-1632. https://doi.org/10.1016/j.theriogenology.2006.02.013

Terrell KA, Wildt DE, Anthony NM, Bavister BD, Leibo SP, Penfold LM, Crosier AE (2012). Different patterns of metabolic cryo-damage in domestic cat (Felis catus) and cheetah (Acinonyx jubatus) spermatozoa. Cryobiology, 64: 110-117. https://doi.org/10.1016/j.cryobiol.2011.12.006

Tsustsui TM, Wada M, Anzai, Hori T (2003). Artificial insemination with epididymal sperm in cats. J. Vet. Med. Sci., 65: 397-399. https://www.jstage.jst.go.jp/article/jvms/65/3/65_3_397/_article/-char/ja/

Tsutsui T, Kuwabara S, Kuwabara K, Kugota Y, Kinjo T, Hori T (2004). Development of spermatogenic function in the sex maturation process in male cats. J. Vet. Med. Sci., 66: 1125–1127. https://www.jstage.jst.go.jp/article/jvms/66/9/66_9_1125/_article/-char/ja/ https://doi.org/10.1292/jvms.65.397

Turri F, Madeddu M, Gliozzi TM, Gandini G, Pizzi F (2012). Influence of recovery methods and extenders on bull epididymal spermatozoa quality. Reprod. Domest. Anim., 47: 712-717. https://doi.org/10.1111/j.1439-0531.2011.01948.x

Vansandt LM, Bateman HL, Miller AG, Herrick JR, Moresco A, González R (2021). Cross-species efficacy of a chemically-defined, soy lecithinbased cryomedium for semen banking in imperiled wild felids. Theriogenology, 159: 108-115. https://doi.org/10.1016/j.theriogenology.2020.10.021

Vizuete G, Jiménez E, Agüera EI, Pérez-Marín CC (2014). Impact of ultra-rapid freezing on the motility, morphology, viability and acrosome integrity of epididymal cat sperm diluted in sucrosebased extenders. Reprod. Domest. Anim., 49: e5-e8. https://doi.org/10.1111/rda.12253

Woodruff K, Rigdon-Brestle K, Bushby PA, Wills R, Huston C (2015). Scrotal castration versus prescrotal castration in dogs. Vet Med, 110:131-135. http://veterinarymedicine.dvm360.com/scrotal-castration-versus-prescrotal-castration-dogs

Yani A, Nuryadi PT (2001). Pengaruh tingkat substitusi santan kelapa pada pengencer tris dan waktu penyimpanan terhadap kualitas semen kambing peranakan ettawa (PE)”. Jurnal Biosain, 1, 23-29.

Zambelli D, Cunto M (2006). Semen collection in cats: techniques and analysis. Theriogenology, 66(2), 159-165. https://doi.org/10.1016/j.theriogenology.2006.01.054