Skip to main content
NCBI home page
Poultry Science logoLink to Poultry Science
. 2024 Aug 8;103(11):104190. doi: 10.1016/j.psj.2024.104190

The characteristics of frozen-thawed rooster sperm using various intracellular cryoprotectants

Ahmed M Elomda ⁎, Gamal MK Mehaisen †, Farid KR Stino †, Mohamed F Saad *, Mona M Ghaly †, Agnieszka Partyka ‡,1, Ahmed O Abbas †,§, Farid S Nassar †,§
PMCID: PMC11385514  PMID: 39180781

Abstract

Cryopreservation of rooster semen is essential for conserving genetic resources, genetic improvement, and increasing productivity. However, the nature of avian sperm presents a global issue in ensuring superior frozen semen for artificial insemination. Thus, the present study aimed to evaluate the impact of using dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), and ethylene glycol (EG) as cryoprotectants on post-thawed sperm motility, quality, antioxidant indicators, and fertilizing capacity. Twice a week, fresh semen ejaculates were collected from 15 adult roosters and immediately evaluated to constitute a pool from clean and qualified samples. The pooled semen was further diluted at a ratio of 1:2 (v/v) with an extender and then subjected to a freezing protocol in a liquid nitrogen vapor after adding a cryoprotectant solution containing 6% of either DMA, DMSO, or EG, respectively. After thawing, characteristics of sperm motion, quality, antioxidants, and fertilizing ability were evaluated and compared to fresh and cooled semen as controls. The results demonstrated that semen cooling negatively affected some parameters of sperm motility, quality, antioxidant biomarkers, and fertility. In comparison to the DMSO and EG groups, employing DMA considerably (P < 0.05) raised the percentages of sperm progressive motility, viability, plasma membrane intactness, and DNA integrity. The DMA group showed a significant increase in the catalase and glutathione reduced antioxidant enzyme activity and a reduction in nitric oxide and lipid peroxidation. After artificial insemination, the DMA and DMSO groups exhibited considerably (P < 0.05) better rates of hatchability and fertility than the EG group. It is concluded that freezing extenders containing 6% DMA is better than DMSO or EG to improve the post thaw semen quality and fertility in chickens.

Key words: Rooster, semen freezing, cryoprotectant, sperm quality, fertility

INTRODUCTION

Sperm cryopreservation, among the cryostorage techniques for other germplasm types, remains the most efficient, reliable, and economically assisted reproductive technology for the in vitro conservation and subsequent in vivo reconstitution of poultry genetic resources (Sun, et al., 2022). However, the sudden changes in the temperature during freezing-thawing procedures may induce permanent damage to the avian sperm membrane and structure (Bailey et al., 2000, Purdy and Graham, 2004). This damage, in turn, results in a significant reduction in sperm viability and a substantially low fertilizing ability after employing artificial insemination with frozen-thawed semen (Partyka and Niżański, 2022). In addition, avian sperm has unique characteristics, such as a cylindrical head, long flagellum, little cytoplasm, low antioxidants, minimum mitochondria, and high polyunsaturated fatty acids, which frequently cause various breakage forms and adversely affect the success of the cryopreservation process (Partyka, et al., 2010).

Intracellular cryoprotectants (CPAs) are permeating-cell-membrane compounds that are widely employed in semen extenders to control the ice formation and recrystallization during the freezing/thawing process of rooster sperm (Zong, et al., 2023). Among these intracellular CPAs are ethylene glycol (EG), dimethyl sulfoxide (DMSO), and certain amide compounds like dimethylacetamide (DMA) and dimethylformamide (DMF) (Santiago-Moreno, et al., 2017; Shanmugam et al., 2018). DMSO partially replaces the cell water and rapidly precipitates below the freezing point, allowing the vitrification without the formation of intracellular ice crystals (Rakha, et al., 2018). Moreover, DMSO has a unique combination of hydrophobic (methyl group) and hydrophilic (sulfoxide group) characteristics, which allow DMSO to cross the plasma membrane easily (Best, 2015). DMA is composed of 2 methyl groups bound to one amide group [CH3C(O)N(CH3)2] while it does not have d-orbital functions (Blesbois, 2007). Osuga, et al. (2018) reported that DMA could be potentially effective in sperm cryopreservation because it is normally involved in cellular biological processes and has a high permeability of cell membranes. EG has been also suggested as a practical intracellular CPA due to its characteristics, such as low density, reduced toxicity, fast permeability, and strong binding for the sperm cell membrane (Massip, 2001; Miranda, et al., 2018).

According to (Chaveiro, et al., 2006), DMA is one of the best preferable CPAs for cryopreserving chicken sperm. In previous research, DMA has been incorporated into semen extenders at doses varying between 3-26% to investigate or enhance chicken sperm cryopreservation efficiency. The optimal sperm motility and quality after thawing were obtained at approximately 3-6% DMA concentration across various poultry species (Blanco, et al., 2011; Zaniboni, et al., 2014; Mehaisen, et al., 2022; Hamad, et al., 2023), while higher DMA levels negatively affected the average fertility achieved with cryopreserved chicken semen (Abouelezz, et al., 2015; Abouelezz, et al., 2017; Tang, et al., 2021). DMSO was used at concentrations ranging from 2% to 20% in semen-freezing extenders (Murugesan and Mahapatra, 2020; Rakha, et al., 2020). Previous studies reported that 4-8% of DMSO showed a cryoprotective ability and an appropriate motility, quality, and fertility of frozen/thawed sperm in poultry species (Penfold, et al., 2001; Rakha, et al., 2018; Kumar, et al., 2019). In contrast, EG was used at a concentration ranging from 3% to 16% in the freezing protocols and thawing was applied at 5 or 37°C with various results in motility, viability, and fertility (Mphaphathi, et al., 2016; Miranda, et al., 2018; Olexikova, et al., 2019; Khaeruddin et al., 2020; Rakha, et al., 2020).

In a comparative study between different CPAs, Murugesan and Mahapatra (2020) concluded that fertility rates varied based on the cryoprotectant, diluent, and thawing temperature. They reported that 8% EG and 6% DMF in Lake and Ravie (LR) diluent and thawing at 37°C achieved acceptable fertility rates (48.12 and 30.89%, respectively), compared to less than 1% fertility rate for the other cryoprotectants, such as DMA, DMSO, EG, and DMF, when samples were thawed at 5°C. In another study, Khaeruddin et al. (2020) found that post-thawed sperm quality with 7% DMSO was better than 3, 5, and 7% EG. Moreover, Miranda, et al. (2018) demonstrated that post-thawed sperm motility was improved by combining 8% EG with 5°C thawing compared to 6% DMA, DMF, and 9% MA (methyl acetamide). To our knowledge, such comparisons between intracellular CPA efficacy and mechanism of action for poultry sperm cryopreservation have not been fully understood. Therefore, the current study aimed to investigate the impact of using DMA, DMSO, and EG as intracellular CPAs on the post-thaw sperm motility, quality, antioxidant biomarkers, and fertilizing ability in chickens.

MATERIALS AND METHODS

Animals and Ethical Approval

Fifteen chicken roosters from the Cairo-B2 strain (Hamad, et al., 2023), aged 10 to 12 mo and weighed 3,500 ± 50 g, were used for the present study. All roosters were individually housed in 50 × 50 × 60 cm cages and provided with artificial photoperiods (16L:8D), ad libitum water, and commercial standard diets (2,750 Kcal ME and 14% CP) throughout the experiment. The study was approved by the Institutional Animal Care and Use Committee at Cairo University (CU-II-F-12-20).

Semen Processing and Experimental Protocol

Semen ejaculations were obtained from each male 2 times per week by the dorso-abdominal massage technique (Bakst and Dymond, 2013). The ejaculates were received into sterile tubes and kept in a water bath at 37°C during lab processing. After rapid evaluation, only the samples with normal features and, at least, 4 × 109 sperm/mL concentration and 60% progressive motility were selected to constitute the experimental semen pool. The semen pool was then diluted at a ratio of 1:2 (v/v) with a prewarmed EK extender developed by Lukaszewicz (2002). The diluted semen was equally divided into 3 groups with appropriate labels and then maintained for 1 h in a refrigerator at 5°C. After cooling, a cryoprotectant solution of DMA (Qualikems Fine Chem Pvt. Ltd., Vadodara, India), DMSO (Techno Pharmchem, Haryana, India), or EG (Sigma-Aldrich Inc., St. Louis, MO) was gently added to its respective semen group at a final concentration of 6% and kept at 5°C for 10 min for equilibration. Semen was then uploaded into 250-µL-French straws (Minitube GmbH, Tiefenbach, Germany). The straws (each containing approximately 200 × 106 sperm) were maintained for 10 min at 5 cm above the liquid nitrogen (LN2) vapor within a cork container, then directly plunged into the LN2. The frozen semen was thawed after 3 months of storage inside the LN2 by immersing the straws into a 38°C water bath for 10 seconds. All parameters were evaluated in the fresh diluted semen (Fresh group), immediately after cooling phase of the protocol (Cooled group), and after thawing of the frozen semen with the CPAs (MDA, DMSO, and EG groups).

Sperm Motility Parameters

Ten µL from each treatment group was dropped on a glass slide preheated to 37°C and the sperm motility parameters of 6 random fields (250-300 sperm cells per field) were measured using the computer-assisted sperm analysis (CASA; Sperm Vision™ software, version 3.0 for Win10, Minitube, Tiefenbach, Germany). The CASA settings for roosters are presented in Table 1. The exported parameters by the CASA system included the total (TM, %) and progressive (PM, %) motilities, average-path (VAP, µm/s), curve-line (VCL, µm/s), and straight-line (VSL, µm/s) velocities, straightness (STR, %), linearity (LIN, %), wobble (WOB, %), amplitude of lateral head displacement (ALH, µm), and beat cross frequency (BCF, Hz).

Table 1.

The CASA settings for rooster samples.

Item Specification
CASA software Sperm Vision 3.0 (Minitube)
Microscope model Olympus-BX41 (Tokyo, Japan)
Microscope objective 20 × /0.50 NH negative phase contrast
Temperature control Heated stage unit (37°C)
Working sperm concentration 100 × 106 mL−1
Working semen volume 10 µL
Frame rate 60 s−1
TM VSL > 5 µm/s
PM VAP > 20 µm/s; STR > 80%
STR VSL⁄VAP %
LIN VSL/VCL %
WOB VAP/VCL %
Open in a new tab

CASA, computer assisted sperm analysis; TM, total motility; PM, progressive motility; STR, straightness; LIN, linearity; WOB, wobble; VSL, straight line velocity; VAP, average path velocity; VCL, curved line velocity.

Sperm Quality Parameters

The sperm viability, plasma membrane functionality, and DNA integrity in each treatment group were determined using the techniques outlined in a recent study by Mehaisen et al. (2022) and Hamad, et al. (2023). In summary, semen samples were incubated with Eosin-Nigrosin stain drops (Bio-Diagnostic, Inc., Giza, Egypt) for 30 s. at room temperature then a slide smear from the mixture was scanned for the live (light-unstained) and dead (pink-stained) sperm. The plasma membrane functionality was evaluated by the hypo-osmotic swelling test (HOST). In brief, 10 µL of the semen was added to 100 µL of a hypo-osmotic solution (100 mOsmol/kg) prepared by mixing 1.375 g fructose and 0.75 g sodium citrate dihydrate in a 100-mL distilled water. After incubation in a water bath at 37 °C for 1 h, 20 µL of the mixture was smeared on a preheated slide then sperm were scanned under a phase contrast microscope. Sperm membrane integrity was recognized based on the positive response to the hypo-osmotic solution and featured by swollen or curled tails, while the negative response to the hypo-osmotic solution featured with straight tails. Additionally, the DNA integrity was evaluated in the samples following the methodology of Henkel, et al. (2001). Briefly, semen smears were stained with 5% aniline blue solution (CDH. Ltd., New Delhi, India) acidified with 2% glacial acetic acid for 5 min. As a result, the sperm heads showing intense and very intense aniline blue staining were classified as DNA-fragmented spermatozoa, whereas those stained only weakly or not stained were classified as non-DNA-fragmented spermatozoa. All tests were performed by counting at least 200 sperm cells in each slide using a phase-contrast microscope (Olympus, Tokyo, Japan) at 1,000 × magnification with oil immersion.

Antioxidant Biomarkers Assay

Six semen replicates per treatment group (1 mL semen) were washed twice with PBS, and the semen pellets were collected by centrifugation (1,030 g for 20 min at 4°C). The final pellets were re-suspended in 1 mL PBS supplemented with 4% Triton X-100 and allowed to sit at room temperature for 30 min. Following further centrifugation, the supernatants were collected and snap-frozen at −80°C for further assay. The total protein (TP) content in the samples was first determined using the biuret reaction method, following the colorimetric assay kit's manufacturer's instructions (TP-2020, BioDiagnostic, Inc., Egypt). The antioxidant biomarkers were then measured in the samples, as mentioned below, and the results were normalized per milligram of protein. An automated scanning spectrophotometer (CE1010, Cecil Instruments Limited, Cambridge, United Kingdom) was used to collect the data for all analyses.

Total Antioxidant Capacity (TAC). The TAC was determined using a colorimetric kits (TAC-2513, Bio-Diagnostic, Inc.) according to the procedures described by (Koracevic, et al., 2001). Following the kit's instructions, 20 μL of the sample was mixed with 500 μL of H₂O₂ substrate and incubated at 37°C for 10 min. Then, the mixture was incubated with 500 μL of working chromogen reagent at 37°C for 5 min. The absorbance of the sample (Asample) and blank (Ablank) was measured against distilled water at 505 nm. The TAC was calculated as Ablank–Asample × 3.3.

Superoxide Dismutase (SOD) Activity. The SOD was assessed according to previous methods cited by Elomda, et al. (2018) using a colorimetric assay Kits (SOD-2521, Bio-Diagnostic, Inc.). Briefly, 1 mL of working reagent (1 mL NADH, 1 mL nitro-blue tetrazolium (NBT), and 10 mL phosphate buffer pH 8.5) was mixed well with 100 μL of the control (distilled water) or the sample, and then 100 μL of phenazine methosulphate was added to initiate the reaction. The increase in absorbance at 560 nm over 5 min for the sample (ΔAsample) and the control (ΔAcontrol) was measured at 25°C. The SOD activity was calculated as unit/assay, where unit = (ΔAcontrol–ΔAsample)/ΔAcontrol × 100 × 3.75.

Catalase (CAT) Activity. The CAT was assayed by using a colorimetric assay kits (CAT-2517, Bio-Diagnostic, Inc.), according to previous methods cited by Elomda, et al. (2018). In brief, 50 μL of the sample or the standard was mixed with 100 μL of H₂O₂ diluted by 500 μl phosphate buffer (pH 7.0). After incubation for 1 min at 25°C, 200 μL of chromogen-inhibitor and 500 μL of peroxidase 4-aminoantipyrine enzyme were added to the mixture and incubated for 10 min at 37°C. The same steps were repeated for the sample and standard without addition of H₂O₂ to obtain their blanks. The absorbance of the sample (Asample) against sample blank and the standard (Astandard) against standard blank was read at 510 nm. The CAT activity was calculated as unit per assay, where unit = (Astandard−Asample)/Astandard × 1,000.

Glutathione Reduced (GR) Activity. A colorimetric kits (GR-2511, Bio-Diagnostic, Inc.) was used to measure the GR activity according to the procedures described by Rahman, et al. (2006). Briefly, 500 μL of the sample was mixed well with a reaction reagent containing 1 mL of assay buffer and 100 μL of 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). The DTNB reagent allowed the reduction of glutathione after 5 to 10 min to form a yellow chromophore compound, 5-thionitrobenzoic acid (TNB). The absorbance of the sample against the blank was measured at 405 nm (Asample). The GR activity was calculated as Asample × 2.22.

Lipid Peroxidation (LPO). The determination of LPO level in the sample was achieved indirectly by analyzing malondialdehyde (Elomda, et al., 2018), using a colorimetric assay kits (LPO-2529, Bio-Diagnostic). In brief, 200 µL of the sample or the standard was heated in a boiling water bath with 1 mL of chromogen for 30 min. The absorbance of the sample against blank (Asample) and the standard against distilled water (Astandard) were measured at 534 nm. The LPO level in the sample was calculated as Asample/Astandard × 10.

Nitric Oxide (NO). The method depends on the formation of nitrous acid diazotize sulphanilamide coupled with N-(1-naphthyl) ethylenediamine in the presence of nitrite and acid environment. The levels of NO were measured using a colorimetric kits (NO-2533; BioDiagnostic, Inc.), according to the methods described by Elmetwalli, et al. (2023). In brief, 100 μL of the sample was mixed with 1 mL sulphanilamide and incubated for 5 min. After that, 100 μL of a reagent containing N-(1-Naphthyl)ethylenediamine dihydrochloride was added to the mixture and incubated for 5 min. The absorbance of the sample against blank (Asample) and the standard against standard blank (Astandard) was measured at 540 nm. The NO level in the sample was calculated as Asample/Astandard × 50.

Fertility Trial

A total of 100 Cairo B2 hens (60 wk old) were divided into 5 equal groups for artificial insemination (AI) with the frozen semen of DMA, DMSO, and EG groups, in addition to the fresh and after-cooling semen groups. The AI process was performed 3 times at a 2-d interval in each hen in the late afternoon (4:00–5:00 pm). A pressure to the hen's abdomen was applied to evert the vaginal orifice through the cloaca, then 200 µL of the semen (containing 2 × 108 sperm) was taken by a plastic tube connected to a pipette and deposited 2 to 4 cm into the vaginal orifice concurrently with the release of pressure on the hen's abdomen. Eggs were collected from each hen daily, starting from the second to the seventh day after the first AI, and stored in a cold room at 18°C and 70 to 75% relative humidity. The collected eggs were then incubated for 17 d in a fully automatic multi-stage setter (total capacity of 36,000 eggs, temperature setting at 99.6°F, and humidity setting at 58%) locally manufactured by the Poultry Technical Office (PTO, Alexandria, Egypt). On the 18th day of incubation, eggs were transferred to a single stage hatcher (PTO, capacity of 12,000 eggs every 3 d, setting at 98.5°F and 92°F in dry and wet bulb reading). At the end of egg incubation (21 d), the number of hatched eggs was recorded, while the unhatched eggs were cracked and categorized into pipped eggs, early mortality embryos, late mortality embryos, and infertile eggs.

Statistical Analysis

The SPSS 22.0 software package was used to conduct the statistical tests (IBM Corp., NY, 2013). Shapiro-Wilk's test was initially used to determine if all of the collected data were within the normal distribution. Sperm variables (motility parameters, quality characteristics, and antioxidant biomarkers) were analyzed using a one-way ANOVA, while the fertility data was analyzed using a chi-square test. The Tukey post hoc test was utilized to compare the statistical difference among the treatment groups (fresh, cooled, DMA, DMSO, and EG). The significance level was established at P-value < 0.05.

RESULTS

Sperm Motility

The effect of semen cooling and freezing with DMA, DMSO, and EG on the sperm motility parameters are displayed in Table 2. Compared to the control fresh semen, the TM, PM, VAP, VCL, and VSL were significantly (P < 0.05) decreased after semen cooling. Semen freezing with the CPAs resulted in a significant (P < 0.05) decrease in the TM, PM, VAP, VSL, LIN, WOB and BCF, compared to the fresh and cooled semen groups. It was observed that DMA group was better than DMSO and EG groups in the post-thaw sperm motility, particularly in the PM, VAP, VSL, WOB, and BCF parameters (P < 0.05).

Table 2.

Effect of semen cooling and freezing with DMA, DMSO, and EG as cryoprotectants (CPA) on the motility parameters of rooster sperm.

Parameters Fresh Cooled DMA DMSO EG P-value
TM (%) 81.38 ± 0.702a 74.67 ± 0.628b 59.63 ± 1.295c 55.28 ± 1.328c 55.01 ± 1.266c <0.001
PM (%) 66.40 ± 0.753a 56.39 ± 0.792b 39.68 ± 0.778c 33.92 ± 0.836d 32.00 ± 0.805d <0.001
VAP (µm/s) 74.74 ± 0.998a 65.22 ± 1.069b 59.72 ± 0.884c 56.69 ± 0.754cd 56.26 ± 0.661d <0.001
VCL (µm/s) 118.56 ± 1.353a 104.53 ± 1.232b 104.27 ± 1.827b 104.13 ± 1.550b 104.13 ± 1.708b <0.001
VSL (µm/s) 47.80 ± 0.916a 42.55 ± 0.899b 37.93 ± 0.702c 36.09 ± 0.542cd 35.21 ± 0.396d <0.001
STR (%) 63.43 ± 0.696 64.68 ± 0.575 63.00 ± 0.006 63.00 ± 0.006 62.00 ± 0.006 0.182
LIN (%) 39.80 ± 0.675a 40.18 ± 0.638a 36.00 ± 0.007b 34.00 ± 0.006b 34.00 ± 0.006b <0.001
WOB (%) 62.63 ± 0.427a 61.82 ± 0.513a 57.00 ± 0.005b 54.00 ± 0.005c 54.00 ± 0.005c <0.001
ALH (µm) 4.79 ± 0.060ab 4.61 ± 0.047b 4.81 ± 0.045ab 4.85 ± 0.058a 4.84 ± 0.046a 0.024
BCF (Hz) 29.51 ± 0.196a 28.88 ± 0.251a 26.29 ± 0.263b 25.89 ± 0.329bc 25.09 ± 0.224c <0.001
Open in a new tab

Data are presented as means ± standard error (SE). Means with uncommon superscripts, within the same row, significantly differ at P<0.05. Fresh: fresh semen as control; Cooled: semen after cooling without CPAs: DMA: semen frozen with dimethylacetamide; DMSO: semen frozen with dimethyl sulfoxide; EG: semen frozen with ethylene glycol. TM: total motility; PM: progressive motility; VAP: average path velocity; VCL: curved line velocity; VSL: straight line velocity; STR: straightness; LIN: linearity; WOB: wobble; ALH: amplitude of lateral head displacement; BCF: beat cross frequency.

Sperm Quality Parameters

The effect of semen cooling and freezing with DMA, DMSO, and EG on the sperm quality parameters are illustrated in Figure 1. The sperm viability and plasma membrane integrity were significantly (P < 0.05) decreased after cooling and freezing compared to the fresh semen. Within the CPAs groups, the highest sperm viability and plasma membrane integrity was recorded (P < 0.05) in the DMA group followed by the DMSO group and then the EG group (Figures 1A and 1B). In contrast, the sperm DNA fragmentation was significantly (P < 0.05) higher in the EG group than the DMSO group in comparison with the DMA, cooled and fresh groups (Figure 1C).

Figure 1.

Figure 1

Open in a new tab

Effect of semen cooling and freezing with DMA, DMSO, and EG as cryoprotectants (CPA) on the quality parameters of rooster sperm. Viability (panel A), Plasma membrane integrity (panel B), and DNA fragmentation (panel C). Bars express means ± standard error (SE). Bars with different superscripts represent significant differences (P < 0.05). Fresh: fresh semen as control; Cooled: semen after cooling without CPAs: DMA: semen frozen with dimethylacetamide; DMSO: semen frozen with dimethyl sulfoxide; EG: semen frozen with ethylene glycol.

Sperm Antioxidant Biomarkers

The effect of semen cooling and freezing with DMA, DMSO, and EG on the sperm antioxidant biomarkers are shown in Figure 2. The results indicated that sperm TAC was significantly (P < 0.05) decreased after cooling and freezing with CPAs compared to the fresh semen (Figure 2A). No significant differences were observed in the antioxidant enzymes SOD, CAT, and GR activity after cooling compared to fresh semen. After semen freezing and thawing, the antioxidant enzymes activity significantly (P < 0.05) decreased compared to the fresh and cooled semen. However, the CAT and GR activity was better (P < 0.05) in the DMA group than the DMSO and EG groups (Figures 2C and 2D). On the contrary, the oxidative substrates, LPO and NO levels, were significantly (P < 0.05) higher in the DMSO and EG groups, compared to the DMA, cooled, and fresh semen groups (Figures 2E and 2F).

Figure 2.

Figure 2

Open in a new tab

Effect of semen cooling and freezing with DMA, DMSO, and EG as cryoprotectants (CPA) on the antioxidant biomarkers of rooster sperm. TAC: total antioxidant capacity (panel A), SOD: superoxide dismutase (panel B), CAT: catalase (panel C), GR: glutathione reduced (panel D), LPO: lipid peroxidation (panel E), and NO: nitric oxide (panel F). Bars express means normalized per mg protein ± standard error (SE). Bars with different superscripts represent significant differences (P < 0.05). Fresh: fresh semen as control; Cooled: semen after cooling without CPAs: DMA: semen frozen with dimethylacetamide; DMSO: semen frozen with dimethyl sulfoxide; EG: semen frozen with ethylene glycol.

Sperm Fertility Evaluation

The results of fertility trial after AI with fresh, cooled, and frozen semen of cryoprotectant groups are shown in Table 3. It was found that AI with the cooled semen significantly (P < 0.05) decreased the percentage of fertile eggs and increased the late embryo death rate, compared to the results obtained by fresh semen. After AI with the frozen semen, a significant (P < 0.05) decrease in egg fertility and hatchability occurred in the DMA and DMSO groups in comparison with the fresh and cooled semen groups, while no fertile eggs were obtained from the EG group. It was also observed that early death embryo occurred only in 25% of the fertile eggs obtained from the DMSO group, compared to the other groups (P < 0.05).

Table 3.

The fertility traits of rooster sperm after artificial insemination with semen fresh, cooled, and frozen with DMA, DMSO, and EG as cryoprotectants (CPA).

Parameters Fresh Cooled DMA DMSO EG P-value
Incubated eggs 84 81 85 85 80
Fertile eggs1 60 (71.4%)a 43 (53.1%)b 14 (16.5%)c 8 (9.4%)cd 0 (0.0%)d < 0.001
Hatched eggs2 58 (96.7%)a 38 (88.4%)a 13 (92.9%)a 6 (75.0%)a 0 (0.0%)b < 0.001
Pipped eggs2 2 (3.3%) 0 (0.0%) 1 (7.1%) 0 (0.0%) 0 (0.0%) 0.549
Early embryo death2 0 (0.0%)b 0 (0.0%)b 0 (0.0%)b 2 (25.0%)a 0 (0.0%)b < 0.001
Late embryo death2 0 (0.0%)b 5 (11.6%)a 0 (0.0%)b 0 (0.0%)b 0 (0.0%)b 0.033
Open in a new tab
1

Calculated as a percentage of total incubated eggs.

2

Calculated as percentages of fertile eggs. Means with uncommon superscripts, within the same row, significantly differ at P < 0.05. Fresh: fresh semen as control; Cooled: semen after cooling without CPAs: DMA: semen frozen with dimethylacetamide; DMSO: semen frozen with dimethyl sulfoxide; EG: semen frozen with ethylene glycol.

DISCUSSION

Most sperm cryopreservation protocols in chicken and other species include a cooling phase before freezing application to equilibrate the sperm with low temperature and keep survive during the procedure (Blank, et al., 2021). However, chicken spermatozoa in the present study were susceptible to the cooling process (one hour in a refrigerator at 5°C before freezing) and various parameters of sperm motility, quality, antioxidant, and fertility were remarkably decreased in the cooled semen compared to the fresh semen. This may be due to the methodology of cooling rate in our study which was uncontrolled inside the common refrigerators (Ashrafi et al., 2011).

Sperm motility is a key factor in evaluating the quality of sperm, fertilizing capacity, and cryopreservation efficiency (Lange-Consiglio, et al., 2013). The current study showed that the post-thaw PM of rooster sperm were substantially improved by using the DMA rather than DMSO or EG at a constant level of 6% in the freezing extenders. These results are comparable to those of Murugesan and Mahapatra (2020), who found that post-thawed semen cryopreserved with DMA had a significantly higher progressive motility than semen cryopreserved with DMSO. Several researchers suggest different effects for CPAs on motility when implementing different thawing temperatures. For example, Miranda, et al. (2018) found that TM and PM did not differ between 6% DMA and 8% EG when thawing the rooster sperm at 37°C; whereas better motility was obtained with EG after thawing at 5°C. Moreover, our data displayed a better VAP, VSL, WOB, and BCF when using DMA than DMSO and EG.

One of the most detrimental factors during semen manipulation for cryopreservation is the excessive production of reactive oxygen species (ROS), free radicals, and reactive nitrogen species (RNS) (Sicherle, et al., 2011; Doshi, et al., 2012). The presence of such products in a high concentration may increase sperm abnormality and damage and, subsequently, induce a dramatic decrease in sperm motility and viability (Vignini, et al., 2006). The current study showed that sperm frozen with DMA significantly had lower lipid peroxidation and nitric oxide levels in comparison with DMSO and EG groups. The excessive levels of NO itself may be linked directly to the oxidation of polyunsaturated fatty acids (PUFA) in the sperm membrane, facilitating the sperm LPO (Makker, et al., 2009; Bain, 2010). High ROS production in the sperm may accompany the high LPO and NO levels in the DMSO and EG groups (Partyka, et al., 2012), and as a result the ATP production was impeded in the sperm (Guthrie and Welch, 2012). In contrast, the enhanced CAT and GR antioxidant enzyme activity may also contribute to better sperm quality and motility in the DMA-sperm group, compared to the DMSO and EG groups, by ameliorating the negative effects of LPO and NO after thawing. These findings agree with previous studies reporting that sperm motility and quality dramatically decreased in the presence of high LPO (Mussa, et al., 2020; Hamad, et al., 2023) or NO (Ortega Ferrusola, et al., 2009; de Andrade, et al., 2018) contents.

The present study showed that sperm viability and plasma membrane integrity were higher in the DMA group followed by DMSO and EG groups. This result agrees with Murugesan and Mahapatra (2020) and Woelders, et al. (2006) who reported a higher sperm viability in the DMA group than DMSO and EG. Gerzilov (2010) documented that the motility and viability of post-thawing Muscovy spermatozoa were lower by using EG compared to DMSO. The improvement in sperm quality parameters in the DMSO group compared to EG may be due to specific features of the EG. It was reported that EG has a high penetrating ability into the cell (Seshoka, et al., 2016) and high toxicity in warm conditions like that induced by the thawing procedure (Bhattacharya, 2018), and thus it may cause a sustainable cell membrane damage. Moreover, prior research demonstrated that EG has a lower molecular weight than DMSO and can penetrate the sperm plasma membrane more quickly, harming the sperm during equilibration, freezing, and thawing procedures (Gilmore, et al., 2000; Awad, 2011). Similarly, the highest DNA integrity was counted in the sperm of the DMA group followed by the DMSO group, while a notable fragmentation was observed in the EG group (Figure 1C).

The reduction of rooster fertility in all treatment groups in the present study may be due to the aging (Fouad, et al., 2020), strain (Ayeneshet, et al., 2024), and steps of the cryopreservation technology, including semen dilution, cryoprotectants, equilibration time, packaging types, and freezing and thawing rates (Zong, et al., 2023). However, the best fertilization and hatchability rates were obtained in the DMA and DMSO groups compared to the EG group (Table 3). According to Murugesan and Mahapatra (2020), cryopreserved semen containing 8% EG and thawed at 37°C produced 18 to 48% fertility, but semen frozen with 6% DMA, 2% DMSO, and 8% EG produced no viable eggs when thawed at 5°C. It is well-recognized that the creation of ice crystals, ROS, and cryoprotective agents can all harm DNA during the freezing/thawing process (Velarde et al., 2023). Despite having damaged DNA, several studies have indicated that cryoprotective agents (CPA) containing an amide group provide greater DNA protection compared to other agents like glycerol and DMSO (Figueroa, et al., 2016; Perry, et al., 2019). They also attributed this amide-CPAs priority to the low molecular weight and viscosity of amides, which in turn minimize the cell damage induced by osmotic stress. However, previous studies (Pérez-Cerezales, et al., 2011; Gallego, et al., 2013) reported that although fertilized eggs could be obtained from DNA-fragmented sperm, such fertilized eggs almost fail to complete the embryo development and hatch. Moreover, Pérez-Cerezales, et al. (2010) demonstrated that a reduction in viable sperm cells and an increase in DNA fragmentation can lead to a decrease in sperm fertilization capacity and a higher likelihood of fertilization by sperm with damaged DNA, resulting in more non-viable embryos. These findings may interpret the non-fertile eggs obtained in the EG group and the high embryonic death in the DMSO group, compared to the results obtained in the DMA group.

CONCLUSIONS

The current study revealed that semen cooling negatively affected some parameters of sperm motility, quality, antioxidant biomarkers, and fertility. After freezing and thawing protocol, the quality and DNA integrity of sperm were superior in the DMA group compared to the DMSO and EG groups. Furthermore, fertility and hatchability rates exhibited a greater magnitude in the DMA and DMSO groups compared to the EG group. The post-thawed sperm cells in the DMA group exhibited superior antioxidant defense status, as shown by higher levels of CAT and GR, and lower levels of LPO and NO, compared to DMSO and EG groups. Therefore, including DMA in the semen extender for freezing might be deemed a suitable cryoprotectant, resulting in satisfactory outcomes in terms of post-thawed sperm quality and fertility.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

The authors acknowledge the staff of the Egyptian Academy of Scientific Research and Technology (ASRT) for their administrative, technical, and financial support during this study. The APC/BPC is financed by Wroclaw University of Environmental and Life Sciences.

REFERENCES

  1. Abouelezz F., Castaño C., Toledano-Díaz A., Esteso M., López-Sebastián A., Campo J., Santiago-Moreno J. Effect of the interaction between cryoprotectant concentration and cryopreservation method on frozen/thawed chicken sperm variables. Reprod. Domest. Anim. 2015;50:135–141. doi: 10.1111/rda.12464. [DOI] [PubMed] [Google Scholar]
  2. Abouelezz F., Sayed M., Santiago-Moreno J. Fertility disturbances of dimethylacetamide and glycerol in rooster sperm diluents: discrimination among effects produced pre and post freezing-thawing process. Anim. Reprod. Sci. 2017;184:228–234. doi: 10.1016/j.anireprosci.2017.07.021. [DOI] [PubMed] [Google Scholar]
  3. Ashrafi I., Kohram H., Naijan H., Bahreini M. Effect of controlled and uncontrolled cooling rate on motility parameters of cryopreserved ram spermatozoa. BMC Research Notes. 2011;4:547. doi: 10.1186/1756-0500-4-547. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  4. Awad M.M. Effect of some permeating cryoprotectants on CASA motility results in cryopreserved bull spermatozoa. Anim. Reprod. Sci. 2011;123:157–162. doi: 10.1016/j.anireprosci.2011.01.003. [DOI] [PubMed] [Google Scholar]
  5. Ayeneshet B., Taye M., Esatu W., Tsefa A. Comparative analysis of semen quality and fertility in diverse rooster breeds: a systematic review. World’s Poultry Science Journal. 2024;80:1–29. [Google Scholar]
  6. Bailey J.L., Bilodeau J.F., Cormier N. Semen Cryopreservation in Domestic Animals: A Damaging and Capacitating Phenomenon. J Androl. 2000;21:1–7. doi: 10.1002/j.1939-4640.2000.tb03268.x. [DOI] [PubMed] [Google Scholar]
  7. Bain J. Testosterone and the aging male: to treat or not to treat? Maturitas. 2010;66:16–22. doi: 10.1016/j.maturitas.2010.01.009. [DOI] [PubMed] [Google Scholar]
  8. Bakst M., Dymond J. Artificial insemination in poultry, Success in Artificial Insemination-Quality of Semen and Diagnostics Employed. IntechOpen; , United Kingdom: 2013. pp. 722–723. [Google Scholar]
  9. Best B.P. Cryoprotectant toxicity: facts, issues, and questions. Rejuvenation Research. 2015;18:422–436. doi: 10.1089/rej.2014.1656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bhattacharya S. Cryopreservation Biotechnology in Biomedical and Biological Sciences. IntechOpen; United Kingdom: 2018. Cryoprotectants and their usage in cryopreservation process; pp. 7–19. [Google Scholar]
  11. Blanco J.M., Long J.A., Gee G., Wildt D.E., Donoghue A.M. Comparative cryopreservation of avian spermatozoa: Benefits of non-permeating osmoprotectants and ATP on turkey and crane sperm cryosurvival. Anim. Reprod. Sci. 2011;123:242–248. doi: 10.1016/j.anireprosci.2010.12.005. [DOI] [PubMed] [Google Scholar]
  12. Blank M.H., Ruivo L.P., Novaes G.A., Lemos E.C., Losano J.D., Siqueira A.F. Assessing different liquid-storage temperatures for rooster spermatozoa. Animal Reproduction Science. 2021;233:106845. doi: 10.1016/j.anireprosci.2021.106845. [DOI] [PubMed] [Google Scholar]
  13. Blesbois E. Current status in avian semen cryopreservation. World's Poult. Sci. J. 2007;63:213–222. [Google Scholar]
  14. Chaveiro A., Machado L., Frijters A., Engel B., Woelders H. Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports. Theriogenology. 2006;65:1875–1890. doi: 10.1016/j.theriogenology.2005.10.017. [DOI] [PubMed] [Google Scholar]
  15. de Andrade A.F., Arruda R.P., Torres M.A., Pieri N.C., Leite T.G., Celeghini E.C.C., Oliveira L.Z., Gardés T.P., Bussiere M.C.C., Silva D.F. Nitric oxide in frozen-thawed equine sperm: effects on motility, membrane integrity and sperm capacitation. Anim. reprod. sci. 2018;195:176–184. doi: 10.1016/j.anireprosci.2018.05.022. [DOI] [PubMed] [Google Scholar]
  16. Doshi S.B., Khullar K., Sharma R.K., Agarwal A. Role of reactive nitrogen species in male infertility. Reprod. Biol. Endocrinol. 2012;10:109. doi: 10.1186/1477-7827-10-109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Elmetwalli A., Hashish S.M., Hassan M.G., El-Magd M.A., El-Naggar S.A., Tolba A.M. Modulation of the oxidative damage, inflammation, and apoptosis-related genes by dicinnamoyl-L-tartaric acid in liver cancer. Naunyn Schmiedebergs Archives of Pharmacology. 2023;396:3087–3099. doi: 10.1007/s00210-023-02511-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Elomda A.M., Saad M.F., Saeed A.M., Elsayed A., Abass A.O., Safaa H.M., Mehaisen G.M.K. Antioxidant and developmental capacity of retinol on the in vitro culture of rabbit embryos. Zygote. 2018;26:326–332. doi: 10.1017/S0967199418000308. [DOI] [PubMed] [Google Scholar]
  19. Figueroa E., Valdebenito I., Merino O., Ubilla A., Risopatrón J., Farias J.J. Cryopreservation of Atlantic salmon Salmo salar sperm: effects on sperm physiology. J. Fish Biol. 2016;89:1537–1550. doi: 10.1111/jfb.13052. [DOI] [PubMed] [Google Scholar]
  20. Fouad A.M., El-Senousey H.K., Ruan D., Xia W., Chen W., Wang S. Nutritional modulation of fertility in male poultry. Poultry Science. 2020;99:5637–5646. doi: 10.1016/j.psj.2020.06.083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gallego V., Pérez L., Asturiano J., Yoshida M. Relationship between spermatozoa motility parameters, sperm/egg ratio, and fertilization and hatching rates in pufferfish (Takifugu niphobles) Aquaculture. 2013;416:238–243. [Google Scholar]
  22. Gerzilov V. Influence of various cryoprotectants on the sperm mobility of Muscovy semen before and after cryopreservation. Agric. Sci. Technol. 2010;2:57–60. [Google Scholar]
  23. Gilmore J., Liu J., Woods E., Peter A., Critser J. Cryoprotective agent and temperature effects on human sperm membrane permeabilities: convergence of theoretical and empirical approaches for optimal cryopreservation methods. Human Reprod. 2000;15:335–343. doi: 10.1093/humrep/15.2.335. [DOI] [PubMed] [Google Scholar]
  24. Guthrie H., Welch G. Effects of reactive oxygen species on sperm function. Theriogenology. 2012;78:1700–1708. doi: 10.1016/j.theriogenology.2012.05.002. [DOI] [PubMed] [Google Scholar]
  25. Hamad S.K., Elomda A.M., Sun Y., Li Y., Zong Y., Chen J., Abbas A.O., Stino F.K.R., Nazmi A., Mehaisen G.M.K. The in vitro evaluation of rooster semen pellets frozen with dimethylacetamide. Animals. 2023;13:1603. doi: 10.3390/ani13101603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Henkel R., Menkveld R., Kleinhappl M., Schill W.-B. Andrology: Seasonal changes in human sperm chromatin condensation. J. Assist. Reprod. Genet. 2001;18:371–377. doi: 10.1023/A:1016618405570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Khaeruddin K., Junaedi J., Hastuti H. Cryopreservation of Indonesian native chicken semen by using dimethyl sulfoxide and various level of ethylene glycol as cryoprotectants. Biodiversitas. 2020;21:5718–5722. [Google Scholar]
  28. Koracevic D., Koracevic G., Djordjevic V., Andrejevic S., Cosic V. Method for the measurement of antioxidant activity in human fluids. J. Clin. Pathol. 2001;54:356–361. doi: 10.1136/jcp.54.5.356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kumar K.P., Swathi B., Shanmugam M. Effect of L-glycine and L-carnitine on post-thaw semen parameters and fertility in chicken. Slovak J. Anim. Sci. 2019;52:1–8. doi: 10.1080/00071668.2019.1602249. [DOI] [PubMed] [Google Scholar]
  30. Lange-Consiglio A., Meucci A., Cremonesi F. Fluorescent multiple staining and CASA system to assess boar sperm viability and membranes integrity in short and long-term extenders. Open Vet. J. 2013;3:21–35. [PMC free article] [PubMed] [Google Scholar]
  31. Lukaszewicz E. Cryopreservation of Anser anser L. gander semen. Wydawnictwo Akademii Rolniczej we Wroclawiu; Rozprawy (Poland): 2002. p. 111. [Google Scholar]
  32. Makker K., Agarwal A., Sharma R. Oxidative stress & male infertility. Indian J. Med. Res. 2009;129:357–367. [PubMed] [Google Scholar]
  33. Massip A. Cryopreservation of embryos of farm animals. Reprod. Domestic Anim. 2001;36:49–55. doi: 10.1046/j.1439-0531.2001.00248.x. [DOI] [PubMed] [Google Scholar]
  34. Mehaisen G.M.K., Elomda A.M., Hamad S.K., Ghaly M.M., Sun Y., Li Y., Zong Y., Chen J., Partyka A., Nazmi A. Effect of Dimethylacetamide Concentration on Motility, Quality, Antioxidant Biomarkers, Anti-Freeze Gene Expression, and Fertilizing Ability of Frozen/Thawed Rooster Sperm. Animals. 2022;12:2739. doi: 10.3390/ani12202739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Miranda M., Kulíková B., Vašíček J., Olexiková L., Iaffaldano N., Chrenek P. Effect of cryoprotectants and thawing temperatures on chicken sperm quality. Reprod. Domest. Anim. 2018;53:93–100. doi: 10.1111/rda.13070. [DOI] [PubMed] [Google Scholar]
  36. Mphaphathi M.L., Seshoka M.M., Luseba D., Sutherland B., Nedambale T.L. The characterisation and cryopreservation of Venda chicken semen. Asian Pacific J. Reprod. 2016;5:132–139. [Google Scholar]
  37. Murugesan S., Mahapatra R. Cryopreservation of Ghagus chicken semen: effect of cryoprotectants, diluents and thawing temperature. Reprod. Domest. Anim. 2020;55:951–957. doi: 10.1111/rda.13734. [DOI] [PubMed] [Google Scholar]
  38. Mussa N.J., Ratchamak R., Ratsiri T., Chumchai R., Vongpralub T., Boonkum W., Semaming Y., Chantikisakul V. Lipid peroxidation and antioxidant enzyme activity in fresh rooster semen with normal and low sperm motility. Vet. Integr. Sci. 2020;18:183–192. [Google Scholar]
  39. Olexikova L., Miranda M., Kulikova B., Baláži A., Chrenek P. Cryodamage of plasma membrane and acrosome region in chicken sperm. Anatomia, histologia and embryologia. 2019;48:33–39. doi: 10.1111/ahe.12408. [DOI] [PubMed] [Google Scholar]
  40. Ortega Ferrusola C., Fernández L.G., Macías García B., Salazar-Sandoval C., Morillo Rodríguez A., Rodríguez Martinez H., Tapia J.A., Peña F.J. Effect of cryopreservation on nitric oxide production by stallion spermatozoa1. Biol. Reprod. 2009;81:1106–1111. doi: 10.1095/biolreprod.109.078220. [DOI] [PubMed] [Google Scholar]
  41. Osuga T., Hasegawa T., Aoki M., Fujie M., Nakane T., Asai T. Efficiencies of the cryoprotectants N-Methylacetamide, N-Methylformamide and Dimethyl Sulfoxide, and the cell protectants trehalose and hydroxyethyl starch in cryopreservation of swine sperm. Nanomed. Nanotechnol. 2018;3 [Google Scholar]
  42. Partyka A., Łukaszewicz E., Niżański W. Lipid peroxidation and antioxidant enzymes activity in avian semen. Anim. Reprod. Sci. 2012;134:184–190. doi: 10.1016/j.anireprosci.2012.07.007. [DOI] [PubMed] [Google Scholar]
  43. Partyka A., Niżański W. Advances in storage of poultry semen. Anim. Reprod. Sci. 2022;246:106921. doi: 10.1016/j.anireprosci.2021.106921. [DOI] [PubMed] [Google Scholar]
  44. Partyka A., Niżański W., Łukaszewicz E. Evaluation of fresh and frozen-thawed fowl semen by flow cytometry. Theriogenology. 2010;74:1019–1027. doi: 10.1016/j.theriogenology.2010.04.032. [DOI] [PubMed] [Google Scholar]
  45. Penfold L.M., Harnal V., Lynch W., Bird D., Derrickson S.R., Wildt D.E. Characterization of Northern pintail (Anas acuta) ejaculate and the effect of sperm preservation on fertility. Reproduction. 2001;121:267–275. doi: 10.1530/rep.0.1210267. [DOI] [PubMed] [Google Scholar]
  46. Pérez-Cerezales S., Gutiérrez-Adán A., Martínez-Páramo S., Beirão J., Herráez M. Altered gene transcription and telomere length in trout embryo and larvae obtained with DNA cryodamaged sperm. Theriogenology. 2011;76:1234–1245. doi: 10.1016/j.theriogenology.2011.05.028. [DOI] [PubMed] [Google Scholar]
  47. Pérez-Cerezales S., Martínez-Páramo S., Beirão J., Herráez M. Evaluation of DNA damage as a quality marker for rainbow trout sperm cryopreservation and use of LDL as cryoprotectant. Theriogenology. 2010;74:282–289. doi: 10.1016/j.theriogenology.2010.02.012. [DOI] [PubMed] [Google Scholar]
  48. Perry C.T., Corcini C.D., Anciuti A.N., Otte M.V., Soares S.L., Garcia J.R.E., Muelbet J.R.E., Junior A.S.V. Amides as cryoprotectants for the freezing of Brycon orbignyanus sperm. Aquaculture. 2019;508:90–97. [Google Scholar]
  49. Rahman I., Kode A., Biswas S.K. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nature Protocols. 2006;1:3159–3165. doi: 10.1038/nprot.2006.378. [DOI] [PubMed] [Google Scholar]
  50. Rakha B., Ansari M., Akhter S., Akhter A., Blesbois E., Santiago-Moreno J. Effect of dimethylformamide on sperm quality and fertilizing ability of Indian red jungle fowl (Gallus gallus murghi) Theriogenology. 2020;149:55–61. doi: 10.1016/j.theriogenology.2020.03.023. [DOI] [PubMed] [Google Scholar]
  51. Rakha B., Ansari M., Akhter S., Zafar Z., Naseer A., Hussain I., Blesbois E., Santiago-Moreno J. Use of dimethylsulfoxide for semen cryopreservation in Indian red jungle fowl (Gallus gallus murghi) Theriogenology. 2018;122:61–67. doi: 10.1016/j.theriogenology.2018.09.003. [DOI] [PubMed] [Google Scholar]
  52. Santiago-Moreno J., Castaño C., Toledano-Díaz A., Esteso M., López-Sebastián A., Villaverde-Morcillo S., Dávila S., Gil M., Blesbois E. Successful chilling of red-legged partridge (Alectoris rufa) sperm for use in artificial insemination. Poult. Sci. 2017;96:4068–4074. doi: 10.3382/ps/pex176. [DOI] [PubMed] [Google Scholar]
  53. Seshoka M.M., Mphaphathi M.L., Nedambale T.L. Comparison of four different permitting and combination of two best cryoprotectants on freezing Nguni sperm evaluated with the aid of computer aided sperm analysis. Cryobiology. 2016;72:232–238. doi: 10.1016/j.cryobiol.2016.04.001. [DOI] [PubMed] [Google Scholar]
  54. Purdy P.H., Graham J.K. Effect of Adding Cholesterol to Bull Sperm Membranes on Sperm Capacitation, the Acrosome Reaction, and Fertility. Biol Reprod. 2004;71:522–527. doi: 10.1095/biolreprod.103.025577. [DOI] [PubMed] [Google Scholar]
  55. Shanmugam M., Kumar K.P., Mahapatr R.K., Laxmi N.A. Effect of different cryoprotectants on post-thaw semen parameters and fertility in Nicobari chicken. Indian Journal of Poultry Science. 2018;53,:208–211. [Google Scholar]
  56. Sicherle C., Maia M., Bicudo S.D., Rodello L., Azevedo H. Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen supplemented with catalase or Trolox. Small Ruminant Res. 2011;95:144–149. [Google Scholar]
  57. Sun Y., Li Y., Zong Y., Mehaisen G.M.K., Chen J. Poultry genetic heritage cryopreservation and reconstruction: advancement and future challenges. J. Anim. Sci. Biotechnol. 2022;13:1–18. doi: 10.1186/s40104-022-00768-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Tang M., Cao J., Yu Z., Liu H., Yang F., Huang S., He J., Yan H. New semen freezing method for chicken and drake using dimethylacetamide as the cryoprotectant. Poult. sci. 2021;100 doi: 10.1016/j.psj.2021.101091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Velarde J.M.C., Bastos N.M., Carneiro-Leite L., Borges L.P., Vieira E.G., Veríssimo-Silveira R., Ninhaus-Silveira A. Dimethyl acetamide and dimethyl sulfoxide associated at glucose and egg yolk for cryopreservation of Pseudoplatystoma corruscans semen. Neotrop. Ichthyol. 2023;21:1–14. [Google Scholar]
  60. Vignini A., Nanetti L., Buldreghini E., Moroni C., Ricciardo-Lamonica G., Mantero F., Boscaro M., Mazzanti L., Balercia G. The production of peroxynitrite by human spermatozoa may affect sperm motility through the formation of protein nitrotyrosine. Fertility Sterility. 2006;85:947–953. doi: 10.1016/j.fertnstert.2005.09.027. [DOI] [PubMed] [Google Scholar]
  61. Woelders H., Zuidberg C., Hiemstra S. Animal genetic resources conservation in the Netherlands and Europe: poultry perspective. Poult. sci. 2006;85:216–222. doi: 10.1093/ps/85.2.216. [DOI] [PubMed] [Google Scholar]
  62. Zaniboni L., Cassinelli C., Mangiagalli M.G., Gliozzi T.M., Cerolini S. Pellet cryopreservation for chicken semen: Effects of sperm working concentration, cryoprotectant concentration, and equilibration time during in vitro processing. Theriogenology. 2014;82:251–258. doi: 10.1016/j.theriogenology.2014.04.007. [DOI] [PubMed] [Google Scholar]
  63. Zong Y., Li Y., Sun Y., Mehaisen G.M.K., Ma T., Chen J. Chicken sperm cryopreservation: review of techniques, freezing damage, and freezability mechanisms. Agriculture. 2023;13:445. [Google Scholar]

Articles from Poultry Science are provided here courtesy of Elsevier

RESOURCES