The addition of calcium ion chelator,EGTA to thawing solution improves fertilizing ability in frozen–thawed boar sperm

In our previous study, seminal plasma effectively suppressed the induction of sperm to capacitation‐like status and acrosome loss during the thawing process. However, because boar seminal plasma is contaminated with various kinds of bacteria and/or viruses, it is necessary to develop a thawing solution without animal‐derived materials. In this study, we focused on the increase of intracellular Ca²⁺ ([Ca²⁺]_i) in sperm after thawing and the negative effects of sperm qualities. After thawing, the fluorescent intensity of [Ca²⁺]_i indicator, Fluo‐3/AM, and the level of phosphorylated tyrosine residue of protein were increased in the sperm. Next, we investigated whether the addition of Ca²⁺ chelators (ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA)) improved post‐thawed sperm motility. When the frozen–thawed sperm were treated with 6 mmol/L EDTA + 6 mmol/L EGTA, sperm motility was significantly increased as compared with control (6 mmol/L EDTA alone) at all incubation periods (P < 0.05). The combinational treatment significantly suppressed the elevation of [Ca²⁺]_i and the tyrosine phosphorylation, which improved the acrosomal status and fertilizing ability in vitro. Furthermore, when the thawing method was applied for artificial insemination, the fertilization rate was significantly higher than control (P < 0.05, 33% vs. 82%). Thus, we conclude that the addition of EDTA + EGTA to thawing solution is a beneficial tool for artificial insemination using frozen–thawed boar sperm.     

On the fertility and survival of deep frozen boar spermatozoa thawed in skim milk

Satisfactory conception rates of deep frozen boar spermatozoa were obtained, with insemination by way of the cervix, after thawing the deep frozen spermatozoa in boar seminal plasma, both in preliminary trials (Crabo & Einarsson 1971, Crabo et al. 1972 b) and in a large field trial (Einarsson et al. 1972). Fertility with pellet frozen boar spermatozoa, thawed without dilution, was reported by Graham et al. (1971 a, b) and Pursel & Johnson (1971).     

Fertility of deep frozen boar spermatozoa: influence of thawing diluents and of boars.

2 insemination trials were conducted to assess the effect of different thawing diluents on deep frozen boar spermatozoa. The diluents used were boar seminal plasma, protein free seminal plasma, isotonic glucose solution, and OLEP (fructose, sodium pyruvate, CaC12, MgC12, KCL, NaC1, and NaHC03). The electrolyte levels and pH and osmotic pressure of the latter are similar to those of boar seminal plasma. 139 gilts were inseminated. The percentage of fertilized ova was similar for OLEP, protein free seminal plasma, and boar seminal plasma. However, thawing in isotonic glucose solution gave markedly poorer results. There were also differences in the fertility of spermatozoa from the different boars, though these were independent of the effects of the various thawing diluents. The possible effects of the thawing diluents are discussed. The need for methods of selecting boars with spermatozoa of good freezing quality is emphasized.     

Addition of oxytocin to semen extender improves both sperm transport to the oviduct and conception rates in pigs following AI

Oxytocin (OXT) contained in boar semen is known to produce uterine contraction; therefore, we hypothesized that the co‐injection of OXT with sperm would improve artificial insemination (AI) using liquid or frozen‐thawed boar sperm. We initially examined whether OXT added to semen extender improved sperm transport to the oviduct. Although the addition of OXT did not affect the fresh or frozen‐thawed sperm motility or acrosomal integrity, it significantly increased the number of sperm in the oviduct at 6 h after AI injection with OXT, as compared with the control (P < 0.05). Moreover, some sperm were observed in the sperm reservoir of the isthmus in the OXT treatment group, whereas few sperm were observed in the control. When OXT was added to the semen extender immediately prior to AI, the conception rates were significantly higher in both fresh semen and frozen‐thawed semen than in the control group (P < 0.05: liquid, 87.5% vs. 70.5%; frozen‐thawed, 89.8% vs. 75.0%). From these results, we concluded that the addition of OXT to the semen extender assisted in sperm transportation from the uterus to the oviduct, which resulted in improved reproductive performance.     

Fertility of deep frozen boar spermatozoa at various intervals between insemination and induced ovulation: influence of boars and thawing diluents.

The fertilizing ability of deep frozen boar spermatozoa was assessed after artificial insemination at various intervals after human chorionic gonadotropin (HCG) induced ovulation in gilts. Boar seminal plasma and OLEP were used as thawing diluent agents. The fertilization rates were similar when insemination was performed 2 or 6 hours before the time of estimated ovulation (40 hours post-HCG administration). However, fertility rates markedly declined when insemination was performed earlier than 6 hours before expected ovulation. In inseminations performed 2 hours before ovulation, the fertility rate was higher with specimens treated with boar seminal plasma diluent than with OLEP. As the difference between the time of insemination and that of ovulation increased, the difference in fertilizing ability of spermatozoa from the 2 boars used also increased. The results show that spermatozoa which have a low resistance to freezing-thawing also have a diminished fertile life in the female genital tract.     

Effect of Post‐Thaw Addition of Seminal Plasma on Motility,Viability and Chromatin Integrity of Cryopreserved Donkey Jack (Equus asinus) Spermatozoa

Pregnancy rates in donkeys after artificial insemination with cryopreserved semen are still low, compared to the horse species. Addition of autologous seminal plasma to frozen‐thawed semen appeared to improve pregnancy rates. The aims of this study were to evaluate (1) sperm motility and plasma membrane integrity after thawing (T0) and after one and 2 h (T1 and T2) of post‐thaw incubation in either 0% (SP0) or 70% (SP70) autologous seminal plasma and (2) sperm motility, plasma membrane integrity and DNA quality (%COMP‐αt) after thawing (T0) and after 2 and 4 h (T2 and T4) of post‐thaw incubation in either 0% (SP0), 5% (SP5) or 20% (SP20) homologous seminal plasma. In experiment 1, seminal plasma decreased total and progressive sperm motility and plasma membrane intact spermatozoa immediately after dilution and at all following time points (p < 0.05). In experiment 2, total and progressive motility did not differ between treatments immediately after dilution and between SP0 and SP5 at T2, while they were lower in both SP5 and SP20 than in SP0 at T4. Plasma membrane intact sperm cells did not differ between SP0 and SP5 and were lower in SP20 at all time points. DNA quality was not affected by treatment immediately after dilution and was significantly worse for SP20 after 4 h of incubation (p < 0.05). The post‐thaw addition of seminal plasma at the tested concentrations did not improve donkey frozen semen characteristics in vitro over time.     

Influence of boars on the relationship between fertility and post thawing sperm quality of deep frozen boar spermatozoa.

The possibility of selecting boars for deep freezing of spermatozoa was evaluated by tests of frozen-thawed spermatozoa. 20 of 31 ejaculates were used for artificial insemination of 37 gilts. Boar seminal plasma and OLEP were used as thawing diluents. The thermoresistance test and the extracellular concentration of aspartate aminotransferase (ASAT) were used as indicators of fertility. Spermatozoa from 4 boars were utilized. 1 of the boars appeared to be of superior fertility, while the spermatozoa from another was infertile. The latter animal had been used for fresh artificial insemination trials, and had shown higher pregnancy rates than the average. Samples from this boar exhibited the lowest degree of motility after 3 hours storage at 37 degrees C, the greatest relative decrease of motility during the thermoresistance test, the greatest release of ASAT after thawing by OLEP, and greatest relative release of ASAT. These variations were statistically significant (p less than .05). The results show that there is a possibility of detecting boars with spermatozoa with potential low freezability.     

牛精子冷冻损伤研究进展

人工授精技术是目前通过冷冻精液进行牛遗传改良而应用最为广泛的生物技术。虽然如此,由于精液产品的质量而导致人工授精的遗传影响被限制,在精液的冷冻－解冻过程中40%~50%的活精子失去完整性或功能受到损伤。作者在参阅国内外相关文献的基础上,通过阐述冷冻－解冻对精子细胞的损伤作用及机理、精浆对牛冷冻精液质量的影响、冷冻－解冻过程中精子的过氧化损伤,以及冷冻－解冻后精子相关基因与蛋白质的变化等,以期为牛高质量冷冻精液的研究提供一定参考,进一步提升冷冻精液质量。     

The benefits of cooling boar semen in long‐term extenders prior to cryopreservation on sperm quality characteristics

This study investigated the effects of long‐term extenders on post‐thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm‐rich fractions, collected from five boars, were diluted in Androhep^® Plus (AHP), Androstar^® Plus (ASP), Safecell^® Plus and TRIXcell^® Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre‐freeze and frozen‐thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic‐like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing‐thawing. Differences in the pre‐freeze semen were more marked in the sperm motion patterns between the HTs. Pre‐freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO‐PRO‐1^?/PI^?) among the extenders. Post‐thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP‐ and ASP‐extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing‐thawing. In most of the extenders, the incidence of frozen‐thawed spermatozoa with apoptotic‐like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long‐term preservation extenders modulates post‐thaw sperm quality characteristics in an extender‐dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.     

Evaluation of cryopreserved boar semen after supplementation sericin form silkworm (Bombyx mori) in semen extender

Boar cryopreserved semen is scarcely used for artificial insemination due to its quality which is largely reduced by membrane lipid peroxidation. This present study was designed to improve the post‐thawed boar semen quality by determining the optimal level of sericin supplementation (antioxidants) in semen extender. Five levels of sericin supplementation between 0% and 1% (w/v) were examined. Semen was frozen by the liquid nitrogen vapor method, thawed slowly at 5°C for 5 min, and used for the evaluation of sperm quality. The results indicated 0.5%–1% sericin supplementation was more effective on maintenance of sperm viability, acrosome integrity, and mitochondrial functions during freezing–thawing. Moreover, 0.75% sericin supplementation was most protective toward total sperm motility and sperm progressive motility. Additionally, 0.25%–0.75% sericin supplementation significantly suppressed increases in the index of lipid peroxidation. In conclusion, 0.75% sericin is recommended as an alternative component of the freezing extender to improve cryopreserved boar semen. However, further research using AI will be necessary to demonstrate that this indication can be applied to the production of offspring in the farms.     

Effects of supplementation of iodixanol to semen extender on quality and fertilization ability of frozen–thawed Thai native bull sperm

This study investigates the effects of iodixanol supplementation in varied concentrations to Tris egg yolk (TEY) extender on the quality and fertilization ability of frozen–thawed sperm of Thai native bulls. Each ejaculate was divided into four different groups, as follows: sperm were treated with TEY extender (control group) and TEY extender supplemented with three different concentrations of iodixanol (1.25%, 2.50% and 5.00%). Semen straws were frozen in liquid nitrogen vapor. After thawing, sperm motility characteristics, viability, plasma membrane integrity and acrosome integrity were determined. Also, frozen–thawed spermatozoa from all groups were used for in vitro fertilization and artificial insemination (AI) in natural estrus Thai native cows. The results showed that the post‐thaw quality of the 2.50% iodixanol group was superior to the other iodixanol groups (P < 0.05). However, iodixanol had no beneficial effect on post‐thaw sperm in vitro fertilization ability and pregnancy rate after AI (P > 0.05). It can be concluded that the supplementation of 2.50% iodixanol extender significantly improves the progressive motility, viability, plasma membrane integrity and acrosome integrity of cryopreserved semen from Thai native bulls, but it has no beneficial effect on in vitro fertilization ability and pregnancy rate after AI.     

Intrauterine and intravaginal insemination with frozen canine semen using an extender consisting of orvus ES paste-supplemented egg yolk tris-fructose citrate

Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.     

Addition of Seminal Plasma to Post‐thawing Equine Semen: What is the Effect on Sperm Cell Viability?

Effect of seminal plasma addition after thawing on viability or cryocapacitation is not definitively established. This experiment was performed to verify the effect of adding seminal plasma, autologous or homologous (from an animal with good semen freezability). Five ejaculates from each of four stallions with proven fertility were collected and cryopreserved. The semen was subsequently thawed and divided into the following three treatment groups: no seminal plasma addition after semen thawing (NOSP); the addition of homologous seminal plasma after semen thawing (HSP) and the addition of autologous seminal plasma after semen thawing (ASP). The addition of 20% of seminal plasma led to an increase in the cell population that simultaneously show plasma and acrosomal membrane integrity (p < 0.05). The addition of seminal plasma did not alter the total motility, the amount of cells with mitochondrial membrane potential or the sperm velocities (average path velocity, straight-line velocity and curvilinear velocity). However, the beat/cross-frequency, straightness and linearity were reduced in ASP and HSP groups (p < 0.05). Unexpectedly, the addition of homologous seminal plasma reduced the proportion of cells with progressive motility (p < 0.05) and the addition of autologous seminal plasma reduced the amplitude of the lateral head displacement (p < 0.05). Based on the increase in the cell populations that had the plasma and acrosomal membrane integrity simultaneously identified in this study, we proposed that the addition of seminal plasma (autologous or homologous) into post-thawed semen before insemination could increase semen fertility.     

Current challenges in sheep artificial insemination: A particular insight

Ovine artificial insemination (OAI) is not commonly performed because of specific problems related to semen application techniques, leading to highly variable results. The ideal methodology (frozen‐thawed semen/vaginal route) is unfeasible under field conditions due to the cervix morphology of the ewe, which prevents the process of intrauterine insemination necessary to obtain acceptable results. Currently, OAI commercial programmes use superficial cervical insemination, CAI (vaginal), with chilled semen (15°C) and intrauterine insemination, LAI (laparoscopic), with frozen‐thawed semen. The ability to improve upon these contrasting techniques may be derived from examining certain poorly studied factors such as insemination time, productive state of females and alternatives of seminal preservation, some of which we reviewed in this work. This interim solution will remain in use until AI by the vaginal route with frozen‐thawed semen is developed, but it poses new challenges in optimizing the freezing of the sperm and adapting the cervical (CAI) and/or transcervical intrauterine AI (TCAI). In this review, we address the current problems and evaluate their methodological (mechanical) and chemical (dilation) alternatives. Currently, TCAI is a methodologically complex technique with poor fertility results, so further studies are needed to improve the logistics of this procedure and the results of its application.     

Preincubation with green tea polyphenol extract is beneficial for attenuating sperm injury caused by freezing‐thawing in swine

Polyphenols (PFs) extracted from green tea, known to be potent anti‐oxidants, have been reported to be effective in increasing the motility and viability of mammalian sperm, preserved in a liquid form. Therefore, we tested whether PFs might also be effective for maintaining the integrity of frozen‐thawed boar spermatozoa. Ejaculates, collected from Clawn miniature pigs, were diluted in a semen extender containing various amounts of PFs (0, 0.01, 0.05, 0.1 and 0.2% w/v) and then stored at 15°C overnight. The semen samples were processed, using the straw freezing procedure, and then frozen in liquid nitrogen. After rapid thawing at 40°C, the spermatozoa were subjected to several assays to evaluate semen quality. Spermatozoa frozen in a medium containing 0.01% w/v PFs exhibited significantly (P < 0.05) higher degrees of post‐thawed viability and acrosomal integrity than those stored in the absence of PFs. However, no change in the mitochondrial activity was noted between the two groups. The inclusion of 0.01% PFs in the semen extender was significantly (P < 0.05) effective in increasing both the rates of monospermic oocyte formation and of blastocyst formation. These findings indicate that preincubation with the semen extender, containing 0.01% PFs prior to freezing, exerts a protective effect on boar sperm by preventing injuries associated with freezing‐thawing.     

精浆对猪精子冷冻效果影响的研究

为优化猪精子冷冻技术,提高解冻后精子的活力和受精能力,本试验分别以含精浆浓度为10%、20%和30%的冷冻保护剂处理精子,以冷冻前、解冻后精子活力和质膜完整性,解冻后精子进行体外受精(IVF)的卵裂率和囊胚率等作为检测指标,同时以含有卵黄的Tris-柠檬酸－葡萄糖(Tris-citric acid-glucose,TCG)冷冻基础液作为对照研究精浆对猪冷冻精子的保护作用。结果显示,在含有10%精浆浓度的稀释液中,冷冻前质膜完整性,解冻后精子活率、质膜完整性、IVF囊胚率相对于对照组均显著提高(P<0.05);当含有10%精浆的冷冻精液解冻后用于人工授精时,与配母猪妊娠率、窝产仔数、窝产活仔数等仍显著低于鲜精授精组(P<0.05)。上述结果表明,含10%精浆的冷冻保护剂能提高精子的冷冻后活力和IVF胚胎发育率,但用于人工授精配种与鲜精相比还有一定差距。     

Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa

Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaporins (AQPs) 7 and 9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light and electron microscope levels with several commercial primary antibodies and protocols explored AQP location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate), unprocessed, extended, chilled and frozen‐thawed. Although differences in WB and ICC labelling were seen among antibodies, AQP‐7 was conspicuously located in the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid‐piece and principal piece domains in ejaculated spermatozoa. AQP‐9 was mainly localized in the sperm head in both caudal and ejaculated spermatozoa. While unaffected by chilling (+5°C), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP‐7, but not that of AQP‐9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP‐9 but relocated AQP‐7 towards the acrosome. AQP‐7, but not AQP‐9, appears as a relevant marker for non‐empirical studies of sperm handling.     

Effects of Thawing Temperature and Post‐thaw Dilution on the Quality of Cat Spermatozoa

The present study aimed to compare cat sperm quality after thawing using two different temperatures (37 and 70°C) and to investigate the effects of post‐thaw dilution on the sperm quality and longevity of ejaculated cat spermatozoa. Six ejaculates of each of six male cats were collected using an electroejaculator (total 36 ejaculates). The semen was frozen in 0.25‐ml straws using a Tris egg yolk extender containing Equex STM paste. Four straws prepared from each ejaculate were thawed at four different occasions; (i) at 37°C for 15 s, (ii) at 37°C for 15 s and diluted 1 : 2 with Tris buffer (v/v), (iii) at 70°C for 6 s, (iv) at 70°C for 6 s and diluted 1 : 2 with Tris buffer (v/v). The percentages of motile spermatozoa, the scores of progressive motility, the percentages of spermatozoa with intact plasma membrane (using SYBR‐14/EthD‐1 stains) and intact acrosome (using fluorescein isothiocyanate conjugated peanut agglutinin/propidium iodide stains) were evaluated in fresh semen at 0, 2, 4 and 6 h after thawing. The thawing temperature had no effect on any sperm parameters throughout the incubation period (p > 0.05). The dilution after thawing improved sperm motility, progressive motility and acrosome integrity (p < 0.05). The thawing of cat spermatozoa and subsequently diluting with Tris buffer resulted in an immediate (at 0 h) overall (combined over temperature) percentage of motile sperm of 64.8 ± 10.7 (mean ± SD), a score of progressive motility of 4.0 ± 0.5, a percentage of spermatozoa with intact plasma membrane of 64.4 ± 12.1 and intact acrosome of 44.8 ± 20.2. In conclusion, frozen cat semen can be thawed either at 37 or 70°C and post‐thaw dilution is recommended to reduce the toxic effect of some ingredients in the extender during post‐thaw incubation.     

The Effect of Season on Spermatozoa Motility,Plasma Membrane and Acrosome Integrity in Fresh and Frozen–Thawed Semen from Xinong Saanen Bucks

The aim of this study was to evaluate whether the season of ejaculate collection influences seminal quality parameters of pre‐ and post‐freeze–thawing in Xinong Saanen bucks. Ejaculates were collected from eight bucks throughout the four seasons (spring, summer, autumn and winter) in a 12 months’ time period, identified in the Northern Hemisphere. Semen samples were evaluated by the combinations of conventional and Computer‐Assisted Sperm Analysis (CASA) when fresh and after frozen–thawed, respectively. The results clearly demonstrated that season of ejaculate collection influenced (p < 0.05) fresh semen quality. Highest semen quality was observed during autumn. On the contrary, undesirable indices (significantly lower, p < 0.05) were observed in winter as compared with the other remaining seasons. CASA has clearly shown the influences of seasonal variations on semen motility parameters. Furthermore, season of ejaculate collection was also found to influence sperm freezability. Semen characteristics after frozen–thawed followed a similar pattern with that of fresh ejaculate except in spring. The results revealed that sperm quality was higher (p < 0.01) in summer and autumn than in spring and winter. In conclusion, seasonal variation influences semen quality in Xinong Saanen bucks. In addition to summer and autumn, fresh ejaculates in spring can also be successfully used for AI. Sperm from ejaculates collected during summer and autumn are more suitable for cryopreservation. Hence, it is possible to increase the efficiency of goat breeding by manipulating the seasonal variations of semen quality for immediate AI and/or cryopreservation.     

Combined effect of apigenin and ferulic acid on frozen‐thawed boar sperm quality

The main aim of the present study was to evaluate the cryoprotective effect of apigenin (AP) and ferulic acid (FA) on boar sperm during cryopreservation. AP and FA were both demonstrated to be high‐efficiency antioxidants and had not previously been used to protect sperm from cryodamage. As boar sperm is sensitive to oxidative stress, suitable antioxidants are still needed for improving frozen‐thawed sperm quality. With this purpose, semen samples coming from five boars were used in this study. Ejaculates of five boars were mixed and split into 16 aliquots, in which different doses of AP and FA were added separately or together. The motility, the plasma membrane integrity, the mitochondrial activity, the acrosomal integrity, the antioxidase activities and the malondialdehyde concentration of the frozen‐thawed boar sperm were assessed. The results suggested that both AP and FA significantly improved the frozen‐thawed boar sperm quality in all these aspects when they were added to the freezing extender separately, while the highest improvement was recorded when the extender was supplemented with 0.1 mmol/L AP plus 0.15 mmol/L FA. These findings demonstrated that supplementation of freezing extender with both AP and FA had a combined, beneficial effect on frozen‐thawed boar sperm.