Cryopreservation Technologies in Farm Animal Reproduction: Current Status and Future Perspectives

The in vitro production and cryopreservation of mammalian embryos generates reactive oxygen species (ROS) due to conditions of the system that can overcome their antioxidant protection. Resveratrol is an antioxidant used in in vitro systems to improve blastocyst rates, but its effect on antioxidant enzymes such as glutathione (GSH) in embryos produced by in vitro fertilization (IVF) after vitrification has not been reported. The objective of this study was to evaluate the effects of resveratrol on the in vitro maturation medium (IVM) of sheep oocytes (Ovis aries) on the levels of ROS and GSH in embryos produced by IVF subjected to vitrification. Resveratrol was added at 0 µM, 0.25 µM, 0.5 µM, and 1 µM during oocyte in vitro maturation (IVM). Matured oocytes were fertilized with thawed ram sperm. Embryos were cultured in sequential media until blastocysts, were then vitrified for 24 h, and, after heating, they were stained with DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) to determine the presence of ROS and with Cell Tracker Blue^® for the presence of GSH. The quantitative values of ROS and GSH were obtained through the Image J image processor. The results showed that resveratrol increased GSH and decreased ROS production (p < 0.05) in a dose-dependent manner. It is concluded that its use in sheep oocytes during IVM has a beneficial effect on embryos produced by IVF subjected to vitrification by decreasing reactive oxygen species levels and facilitating the generation of embryo antioxidant enzymes like glutathione. Full article

Boar sperm is sensitive to particular conditions during cryopreservation, resulting in an extreme reduction in fertilizing ability due to damage to the sperm membranes. PKMPH contains bioactive peptides that have antioxidant and antimicrobial activities. There is no information on the use of palm-kernel-meal-derived bioactive peptides for boar semen cryopreservation. This study aimed to examine the effects of bioactive peptides from PKMPH on post-thawed boar sperm quality. Boar semen ejaculates (n = 17) were collected and divided into six equal aliquots based on PKMPH concentrations (0, 1.25, 2.5, 5, 10, and 15 µg/mL) in a freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, the frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm motility using a computer-assisted sperm analyzer and for sperm viability, acrosome integrity, mitochondrial function, and lipid peroxidation by measuring the level of malondialdehyde (MDA). The results demonstrate that the supplementation of PKMPH with 2.5 µg/mL afforded superior post-thawed sperm qualities, such as increased total motility, viability, acrosome integrity, and mitochondrial function by 10.7%, 12.3%, 18.3%, and 12.7%, respectively, when compared to the control group. PKMPH at a concentration of 2.5 µg/mL showed the lowest level of MDA (40.6 ± 2.0 µMol/L) compared to the other groups. In conclusion, adding PKMPH peptides at 2.5 µg/mL to the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in higher post-thawed sperm quality. Full article

Thymoquinone nanoparticles (TQNPs) are broadly utilized in numerous pharmaceutical applications. In the present study, we tested the effects of TQNP supplementation on sperm quality and kinematics, acrosome exocytosis, oxidative biomarkers, apoptosis-like and morphological changes of frozen–thawed buffalo sperm, as well as the fertilizing capacity. Semen was collected from buffalo bulls, diluted (1:10; semen/extender), and divided into five aliquots comprising various concentrations of TQNP 0 (CON), 12.5 (TQNP12.5), 25 (TQNP25), 37.5 (TQNP37.5), and 50 (TQNP50) µg/mL, and then cryopreserved and stored in liquid nitrogen (−196 °C). The results revealed that TQNPs (25 to 50 µg/mL) provided the most optimal results in terms of membrane integrity (p < 0.001) and progressive motility (p < 0.01). In contrast, TQNP50 resulted in a greater post-thawed sperm viability (p = 0.02) compared with other groups. The addition of TQNPs to the extender had no discernible effects on sperm morphology measures. Sperm kinematic motion was significantly improved in the TQNP50 group compared to the control group (p < 0.01). TQNPs effectively reduced the content of H₂O₂ and MDA levels and improved the total antioxidant capacity of post-thawed extended semen (p < 0.01). The addition of TQNP significantly increased the number of intact acrosomes (p < 0.0001) and decreased the number of exocytosed acrosomes (p < 0.0001). A significant reduction in apoptosis-like changes was observed in TQNP groups. The non-return rates of buffalo cows inseminated with TQNP50-treated spermatozoa were higher than those in the control group (p < 0.05; 88% vs. 72%). These findings suggested that the freezing extender supplemented with TQNPs could effectively enhance the cryotolerance and fertility of buffalo sperm. Full article

This study aimed to determine the effect of resveratrol and its optimal concentration on the quality of frozen-thawed (FT) boar sperm. Semen ejaculates were obtained from 13 Duroc boars aged between 1.5 and 3 years. The sperm sample was separated into 7 groups based on the concentrations of resveratrol in the freezing extender, which were 0 (control), 25, 50, 75, 100, 125, and 250 µM, respectively. The sperm was frozen using liquid nitrogen vapor and thawed at 50 °C for 12 s. After thawing, total motility, progressive motility, viability, intact acrosomes, mitochondrial membrane potential and level of MDA were assessed. The supplementation of 50–100 µM resveratrol improved the sperm motility and viability of FT sperm in comparison to the control group (p < 0.05). Furthermore, the 50 µM resveratrol group was significantly more protective than the control group in terms of intact acrosome, mitochondrial membrane potential, and level of MDA (p < 0.05). Nonetheless, the detrimental effect of resveratrol was found at a concentration of 250 µM. In conclusion, the addition of 50–100 µM resveratrol to a freezing extender is the optimal concentration for enhancing the quality of cryopreserved boar sperm. Full article

The effectiveness of rabbit-sperm cryopreservation is still below average compared to other domestic species. After the sperm cryopreservation process, post-thawing parameters like motility and membrane integrity are significantly compromised. The use of new extender constituents is an approach that can be used to improve the effectiveness of cryopreservation. Accordingly, we used honey (1.25, 2.5, 5, and 10%), coenzyme Q10 (100 and 200 μM), and β-carotene/α-tocopherol (500 μM/620 μM and 250 μM/310 μM) as candidate components for rabbit-sperm extenders during cryopreservation. Ejaculates from commercial adult rabbit bucks (n = 5) were cryopreserved using conventional freezing. Several post-thawing sperm parameters were assessed, including total motility, membrane integrity, viability, nuclear membrane integrity, acrosome reaction, and mitochondrial membrane potential and activation. Additionally, we performed hormonal analyses of the seminal plasma. Moreover, we analyzed the post-thawing levels of a molecular marker of sperm quality, proAKAP4, which was used in rabbits for the first time. Our findings showed that the 2.5% honey supplementation increased the post-thawing sperm motility (13.75 ± 3.75%) compared to the greater concentrations employed. However, the post-thawing motility was negatively affected by the coenzyme Q10 (0%, in both groups) but was not affected by the β-carotene/α-tocopherol supplementation (22 ± 18.15%, and 11.67 ± 10.17%). In conclusion, the cryopreservation protocols of this study did not help to maintain the sperm parameters after thawing. Further studies are required to identify novel protocols to mitigate the damage caused to rabbit sperm during cryopreservation. Full article

Sperm cryopreservation is an effective technique for conserving animal genetic diversity and transmitting superior genetic backgrounds, maintained via a non-invasive sampling and collection of huge quantities of sperm. Nevertheless, cryopreservation in avian species is not commercially viable because of the rooster sperm’s susceptibility to damage. This study aims to estimate the impact of dimethylacetamide (DMA) as a cryoprotectant at different levels (3%, 6%, or 9%) on the post-thawed sperm quality, motility, antioxidant-biomarkers, and the expression of anti-freeze related genes. Semen samples were collected twice a week from twelve roosters aged 40 wk, weighing 3400 ± 70 g, and belonging to the Cairo-B2 chicken strain. Fresh semen samples were rapidly appraised, pooled, diluted with two volumes of a basic extender, and divided equally into three groups. The diluted groups were chilled at −20 °C for 7 min, then gently supplemented with 3, 6, or 9% pre-cooled DMA and equilibrated at 5 °C for a further 10 min. Semen pellets were formed by pipetting drops 7 cm above liquid nitrogen (LN₂), which were then kept inside cryovials in the LN₂. Thawing was performed 2 months later by taking 3–4 pellets of the frozen semen into a glass tube and warming it in a water bath for 8 s at 60 °C. The results showed that 3% DMA increased the proportion of total motile sperm, progressivity, viability, and plasma membrane integrity (%) compared to the 6% and 9% DMA groups. The lipid peroxidation and antioxidant enzyme activity were improved in the 3% group. At the same time, some anti-freeze-related genes’ (including ras homolog family member A (RHOA), heat shock protein 70 (HSP70), and small nuclear ribonucleoprotein polypeptide A (SNRPA1)) expressions were upregulated within the 3% DMA group relative to other groups. In conclusion, the 3% DMA group maintained higher post-thawed sperm quality than the other tested groups. Full article

Cryo-injury reduces post-thaw semen quality. Extender components play a protective role, but existing experimental approaches do not elucidate interactions among extender components, semen samples, and post-thaw quality. To identify optimal concentrations for 12 extender ingredients, we ran 122 experiments with an I-optimal completely random design using a large dataset from our previous study. We obtained a maximum predicted total motility of 70.56% from an I-optimal design and 73.75% from a Monte Carlo simulation. Individual bull variations were significant and interacted with extenders independently. 67% of bulls reliably preferred extender formulations to reach maximum motility. Multifactor analysis suggests that some antioxidants may offer superior protection over others. Partial least squares path modeling (PLS-PM) found the highest positive loadings for glutathione in the antioxidant class, glycerol in the CPA class, and fructose in the basic compounds class. The optimal ranges for milk, water, and ethylene glycol were extremely narrow. Egg yolk, cholesterol-loaded cyclodextrin, and nerve growth factor had medium-loading impacts. PLS-PM showed that CPA, osmoregulators, and basic components were the most efficient contributors to motility, while the antioxidant and extracellular protectant classes had less efficiency. Thus, ingredients, concentrations, and interactions of extender compounds are critical to extender formulation, especially when using multiple compounds with the same function. Full article

The plasma membrane of spermatozoa plays an important role in the formation and maintenance of many functions of spermatozoa, including during cryopreservation. As a result of chromatographic analysis, the content of lipids and fatty acids in the membranes of spermatozoa of roosters of two breeds was determined under the influence of cryoprotective media containing trehalose LCM-control (0 mM), Treh20 (9.5 mM), and Treh30 (13.4 mM). The use of the cryoprotective diluent Treh20 made it possible to maintain a dynamic balance between the synthesis and degradation of phospholipids and sterols in the plasma membranes of frozen/thawed spermatozoa, close to that of native spermatozoa. This contributed to an increase in the preservation of frozen/thawed spermatozoa membranes from 48.3% to 52.2% in the egg breed and from 30.0% to 35.1% in the meat- and-egg breed. It was also noted that their kinetic apparatus (mobility indicators) remained at the level of 45.6% (egg breed) and 52.4% (meat-and-egg breed). An increase in the concentration of trehalose to 13.4 mM in a cryoprotective diluent for rooster sperm resulted in a decrease in the morphofunctional parameters of frozen/thawed spermatozoa. Full article

During cold storage, boar spermatozoa undergo oxidative stress, which can impair sperm function and fertilizing capacity. The objective of the present study was to assess the effects of Schisandrin B (Sch B) in semen extenders on the quality of boar semen stored at hypothermia. Semen was collected from twelve Duroc boars and diluted in extenders supplemented with different concentrations of Sch B (0 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmol/L, and 40 μmol/L). Here, we demonstrated that 10 μmol/L Sch B provided the best effects on motility, plasma membrane integrity, acrosome integrity, sperm normality rate, average movement velocity, wobbility, mitochondrial membrane potential (MMP), and DNA integrity of sperm. The results of Sch B effects on antioxidant factors in boar sperm showed that Sch B significantly elevated the total antioxidant capacity (T-AOC) and markedly decreased the reactive oxygen species (ROS) and malondialdehyde (MDA) content of sperm. The expression of catalase (CAT) and superoxide dismutase (SOD) mRNA was increased, while the expression of glutathione peroxidase (GPx) mRNA demonstrated no change compared to non-treated boar sperm. Compared to the non-treated group, Sch B triggered a decrease in Ca²⁺/protein kinase A (PKA) and lactic acid content in boar sperm. Similarly, Sch B led to a statistically higher quantitative expression of AWN mRNA and a lower quantitative expression of porcine seminal protein I (PSP-I) and porcine seminal protein II (PSP-II) mRNA. In a further reverse validation test, no significant difference was observed in any of the parameters, including adhesion protein mRNA, calcium content, lactic acid content, PKA, and protein kinase G (PKG) activity after sperm capacitation. In conclusion, the current study indicates the efficient use of Sch B with a 10 μmol/L concentration in the treatment of boar sperm through its anti-apoptosis, antioxidative, and decapacitative mechanisms, suggesting that Sch B is a novel candidate for improving antioxidation and decapacitation factors in sperm in liquid at 4 °C. Full article

Magnetized water is defined as the amount of water that has passed through a magnet. The magnetic field weakens the hydrogen bonds between the water molecules, leading to the magnetized liquid acquiring special characteristics such as easy supercooling and forming smaller ice crystals. We researched the influences of a magnetized freezing extender on cell membrane damage and in vitro fertilization of boar sperm during cryopreservation. The freezing extenders were passed through 0, 2000, 4000, and 6000 gausses (G) of magnetic devices using a liquid cycling pump system and then used for the sperm freezing process. The damage to plasma, acrosomal, and mitochondrial membranes in frozen-thawed spermatozoa was investigated by flow cytometry, and motility was assessed using the CASA system. The fertility of frozen-thawed sperm was estimated using in vitro fertilization. The damage to the membranes was significantly decreased in the magnetized freezing extender by the 6000 G magnetic field compared to that of the control in frozen-thawed sperm, and motility was increased in the 6000 G group. Although there were no significant differences in the cleavage rates of in vitro fertilized oocytes among the treatment groups, the ratio of blastocyst formation increased in the magnetized freezing extender groups compared with that in the control group. The number of blastocysts was significantly higher in the 4000 G group than in the 0 G group. In conclusion, these results suggest that a magnetized freezing extender could improve the freezability of sperm and the development of oocytes fertilized in vitro with frozen-thawed sperm. Full article

In this study, we aimed to determine the benefit of mitoquinone (MitoQ) in rooster semen extenders on sperm quality, motility parameters, antioxidant capacities, and apoptotic changes in post-thawed rooster semen. A total of 85 ejaculates from 18 roosters were collected and then divided into five equal aliquots and cryopreserved in extenders with 1.0% soy lecithin nanoparticles that contained various concentrations of MitoQ (0 nM (M0), 50 nM (M50), 100 nM (M100), 150 nM (M150), and 200 nM (M200)). By using a computer-assisted semen analyzer, sperm motility parameters were assessed after freeze thawing. The M150 group had significantly higher percentages of total motility, progressive motility, viability, acrosome membrane integrity, and mitochondrial activity than the other groups (p < 0.05). Compared to other groups, M100 and M150 groups produced a higher percentage of plasma membrane integrity and ATP contents (p < 0.05). Additionally, the lowest levels of ROS and MDA in spermatozoa were observed in M150 group (p < 0.05), whereas the highest levels of ROS and MDA were observed in sperm in the controls or the M200 group (p < 0.05). Significantly higher values of SOD, GPx, and Cas-3 were found in the M150 group compared to other groups (p < 0.05). Overall, these results demonstrate that MitoQ at 150 nM not only ameliorates post-thawed sperm quality and motility parameters by restoring ATP levels and preventing membrane damage, but also improves redox balance and antiapoptotic activities. Full article

The technology of successful cryopreservation is a very important factor in research and commercial applications. However, the survival and development of the vitrified-thawed (VT) oocytes are lower than those of non-vitrified-thawed (non-VT) oocytes. This study investigated the effect of the addition of hydroxypropyl cellulose (HPC) to a vitrification solution of bovine oocytes. For the vitrification, bovine metaphase II oocytes were pretreated with a solution containing 10% ethylene glycol supplemented with 0, 10, 50, or 100 µg/mL HPC for 5 min, then exposed to a solution containing 30% ethylene glycol supplemented with 0, 10, 50, or 100 µg/mL HPC for 30 sec, and then directly plunged into liquid nitrogen. Oocytes exposed to 0, 10, 50, and 100 µg/mL HPC were named the 0, 10, 50, and 100 HPC groups, respectively. Samples were thawed via sequential incubation in Dulbecco’s phosphate-buffered saline (D-BPS) supplemented with 10% fetal bovine serum and decreasing concentrations of sucrose (1, 0.5, 0.25, and 0.125 M) for 1 min each time. After thawing, VT oocytes were treated at 0.05% hyaluronidase, and cumulus cells were removed by mechanical pipetting. The oocytes were washed with HEPES-buffered Tyrode’s medium and incubated in a droplet of previously cultured in vitro maturation medium for 1 h to recover. The survival rate of the oocytes was significantly higher in the 50 HPC group (84.2%) than in the 0 (75.4%), 10 (80.4%), and 100 (75.5%) HPC groups. The reactive oxygen species (ROS) levels of the non-VT and 50 HPC groups were lower than the 0, 10, and 100 HPC groups. The mRNA levels of proapoptotic genes (Bax) were lower in the non-VT, 0, and 50 HPC groups than in the other groups. The mRNA expression levels of antiapoptotic genes (BCl2) was higher in the non-VT than in the other groups. The mRNA level of a stress-related gene (Hsp70) was lower in the 50 HPC than in the other groups. At day 8, the developmental capacity of embryos obtained via parthenogenetic activation (PA) was determined in the non-VT, 0 HPC, and 50 HPC groups. The cleavage rate of the non-VT group was significantly higher, but the blastocyst development rate and total cell number per blastocyst did not significantly differ between the non-VT and 50 HPC groups. The mRNA levels of proapoptotic genes (Bax and Caspase-3) and a stress-related gene (Hsp70) were higher in the 0 HPC group than in the non-VT and 50 HPC groups. In conclusion, supplementation of vitrification solution with HPC improves the survival rate of VT bovine oocytes and the development capacity of embryos derived from these oocytes via PA. Full article

Thawing protocols have been barely studied, and their modifications may lead to a substantial improvement in post-thawing sperm quality, which could be of great relevance to existing sperm banks, such as those for Iberian pig breeds with varieties in danger of extinction. For that, the study aimed to evaluate different thawing rates and to evaluate modifications in the composition of the thawing extender (basic pH to 8–8.2, incorporation of cyclodextrins loaded with cholesterol [CLC] and the incorporation of ion chelators [EDTA and EGTA]). After thawing, overall sperm motility and kinematic parameters, acrosome status and sperm membrane integrity were evaluated. The most optimal results were obtained with the thawing rate reaching 70 °C for 8 s with the inclusion of 12.5 mg of CLC/500 × 10⁶ spermatozoa in the thawing extender, which showed an improvement compared to the control at 70 °C. In conclusion, to adapt the thawing conditions may be relevant, especially for endangered species or breeds such as some varieties of Iberian pig, since this process could also be used in samples cryopreserved in gene banks. Full article

Cryopreservation is a way to preserve germplasm with applications in agriculture, biotechnology, and conservation of endangered animals. Cryopreservation has been available for over a century, yet, using current methods, only around 50% of spermatozoa retain their viability after cryopreservation. This loss is associated with damage to different sperm components including the plasma membrane, nucleus, mitochondria, proteins, mRNAs, and microRNAs. To mitigate this damage, conventional strategies use chemical additives that include classical cryoprotectants such as glycerol, as well as antioxidants, fatty acids, sugars, amino acids, and membrane stabilizers. However, clearly current protocols do not prevent all damage. This may be due to the imperfect function of antioxidants and the probable conversion of media components to more toxic forms during cryopreservation. Full article

National animal gene banks that are responsible for conserving livestock, poultry, and aquatic genetic resources need to be capable of utilizing a broad array of cryotechnologies coupled with assisted reproductive technologies to reconstitute either specific animals or populations/breeds as needed. This capability is predicated upon having sufficient genetic diversity (usually encapsulated by number of animals in the collection), units of germplasm or tissues, and the ability to reconstitute animals. While the Food and Agriculture Organization of the United Nations (FAO 2012, 2023) developed a set of guidelines for gene banks on these matters, those guidelines do not consider applications and utilization of newer technologies (e.g., primordial germ cells, cloning from somatic cells, embryo transfer, IVF, sex-sorted semen), which can radically change how gene banks collect, store, and utilize genetic resources. This paper reviews the current status of using newer technologies, explores how gene banks might make such technologies part of their routine operations, and illustrates how combining newer assisted reproductive technologies with older approaches enables populations to be reconstituted more efficiently. Full article