The genetic improvement of agriculturally important species and disease control are of fundamental importance to the success of a sustainable agri-food industry. In this sense, artificial insemination (AI) is arguably the most important tool contributing to the advancement of modern animal production. Through AI, 1 ejaculate from a genetically superior male can be used to impregnate multiple females to maximize the distribution of agriculturally favorable genes. As well, AI eliminates physical contact between animals, thus limiting the spread of sexually transmitted diseases. Successful semen cryopreservation enhances these advantages of AI over natural breeding. Long-term storage facilitates semen transport over distances, permits the quarantine of semen, and enables extended use of superior germplasm, even after the sire’s death. For agriculturally important animals, semen cryopreservation is an established industry worldwide, particularly for dairy cattle. Genome resource banking to preserve the biodiversity of endangered species or valuable transgenic lines also would benefit from sperm cryopreservation. For many mammals; however, effective semen cryopreservation is not a reality because a large number of sperm are apparently infertile following freezing and thawing. Compared to fresh, 8 times more cryopreserved bovine sperm were required to achieve equivalent fertilization rates in vivo (Shannon and Vishwanath, 1995). Although AI in pigs with fresh, cooled semen is increasingly popular, the fertility of cryopreserved semen remains about half that of fresh semen (Crabo, 1991). Following either transuterine or oviductal insemination of superovulated ewes, conception rates and percentage of fer-

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.2000.tb03268.x

Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon.

Growing evidence suggests that the generation of reactive oxygen species (ROS) and their detoxification by antioxidants plays a very important role in fertility. However, the relationship between the level of antioxidants in spermatozoa and the decreased fecundity following a freeze/thaw cycle remains poorly understood. We assessed the activities of antioxidant enzymes such as catalase, glutathione peroxidase (GPx), superoxide dismutase (SOD), and levels of reduced/oxidized glutathione (GSH/GSSG) in bovine semen. Sperm cells were isolated using a Percoll gradient to avoid contamination from seminal plasma, cellular debris, and other cell types. We found that bovine spermatozoa are poorly adapted to metabolize the toxic hydrogen peroxide (H(2)O(2)). Indeed, very low levels of GPx and an absence of catalase were observed. We also studied the effect of freezing and thawing bovine spermatozoa in a egg yolk-Tris-glycerol extender (EYTG). Cryopreservation significantly reduced sperm GSH levels by 78% and SOD activity by 50%. We also investigated whether the decrease in GSH level could be linked to oxidative metabolism and found that a greater reduction in intracellular GSH level occurred when fresh sperm cells were incubated in EYTG for 6 hr at 38.5 degrees C under aerobic conditions than when incubated under restricted oxygen availability. Our results strongly suggest the involvement of an oxidative stress during a freeze/thaw cycle and are consistent with the hypothesis that ROS generated during such a cycle are detrimental to sperm function.

Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing.

In the present study, we provide evidence for the production of reactive oxygen species (ROS) during cryopreservation of bovine spermatozoa. Cooling and thawing of spermatozoa cause an increase in the generation of superoxide radicals. Although nitric oxide production remains unaltered during sperm cooling from 22-4 degrees C, a sudden burst of nitric oxide radicals is observed during thawing. Increase in lipid peroxidation levels have been observed in frozen/thawed spermatozoa and appears to be associated with a reduction in sperm membrane fluidity as detected by spin labeling studies. The data presented provide strong evidence that oxygen free radicals are produced during freezing and thawing of bovine spermatozoa and suggest that these reactive oxygen species may be a cause for the decrease in sperm function following cryopreservation. Mol. Reprod. Dev. 59: 451-458, 2001.

Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing.

Current status of sperm cryopreservation: why isn't it better?

Oxidative stress plays a major role in the life and death of mammalian spermatozoa. These gametes are professional generators of reactive oxygen species (ROS), which appear to derive from three potential sources: sperm mitochondria, cytosolic L-amino acid oxidases, and plasma membrane Nicotinamide adenine dinucleotide phosphate oxidases. The oxidative stress created via these sources appears to play a significant role in driving the physiological changes associated with sperm capacitation through the stimulation of a cyclic adenosine monophosphate/Protein kinase A phosphorylation cascade, including the activation of Extracellular signal regulated kinase-like proteins, massive up-regulation of tyrosine phosphorylation in the sperm tail, as well as the induction of sterol oxidation. When generated in excess, however, ROS can induce lipid peroxidation that, in turn, disrupts membrane characteristics that are critical for the maintenance of sperm function, including the capacity to fertilize an egg. Furthermore, the lipid aldehydes generated as a consequence of lipid peroxidation bind to proteins in the mitochondrial electron transport chain, triggering yet more ROS generation in a self-perpetuating cycle. The high levels of oxidative stress created as a result of this process ultimately damage the DNA in the sperm nucleus; indeed, DNA damage in the male germ line appears to be predominantly induced oxidatively, reflecting the vulnerability of these cells to such stress. Extensive evaluation of antioxidants that protect the spermatozoa against oxidative stress while permitting the normal reduction-oxidation regulation of sperm capacitation is therefore currently being undertaken, and has already proven efficacious in animal models.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/mrd.22871

Reactive oxygen species as mediators of sperm capacitation and pathological damage

Poor motility and abnormal acrosomal morphology only partially explain the reduced fertility of cryopreserved bovine spermatozoa. To test the hypothesis that cryopreservation procedures (dilution, cooling, freeze-thaw) induce capacitation in bovine spermatozoa, two experiments were conducted using semen diluted in egg yolk-Tris-glycerol extender (EYTG) (Tris, tris(hydroxymethyl)aminomethane). Capacitation was determined prior to and following incubation with various concentrations of heparin using the chlortetracycline (CTC) fluorescence assay or after preexposure to EYTG using in vitro fertilization (IVF) of bovine cumulus-oocyte complexes (COC) in the absence of heparin. Fresh ejaculates were divided into four treatments and the first was diluted with noncapacitating medium, NCM (+0.3% polyvinyl alcohol (PVA); control), then maintained at 23 degrees C for 4 hours. The remaining semen was diluted with EYTG; the second treatment was held at 4 degrees C (EYTG-4), and the third treatment was held at 23 degrees C (EYTG-23) for 4 hours. The fourth treatment was cooled to 4 degrees C over 4 hours, as per the normal industry protocol, cryopreserved, and thawed (frozen-thawed). After the 4-hour maintenance periods or thawing, all treatments were resuspended either in capacitating medium (CM; +0.6% BSA) for the CTC experiment (n = 3) or in NCM for the IVF experiment (n = 9-11). Prior to incubation in conditions that support capacitation, the percentage of cells exhibiting pattern B (capacitated according to the CTC assay) was similar for all treatments with fresh-extended spermatozoa. Immediately following the addition of heparin (0, 2, or 10 micrograms/ml), three times more frozen-thawed than fresh-extended spermatozoa exhibited pattern B (P < 0.05). After 3 or 6 hours of incubation, however, the percentages of cells displaying pattern B did not differ among treatments. In the absence of heparin, spermatozoa preexposed to EYTG-4 fertilized 2.6x more COC than did control cells (P < 0.001) and 9.2x more than spermatozoa preexposed to EYTG but held at 23 degrees C (EYTG-23; P < 0.0001). No differences were observed among fertilization rates for fresh-extended (EYTG-4) and frozen-thawed spermatozoa. This study provides evidence that premature capacitation occurs in partially (extended and cooled) and fully cryopreserved bovine spermatozoa.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.1997.tb01953.x

Premature Capacitation of Bovine Spermatozoa Is Initiated by Cryopreservation

After ejaculation, mammalian spermatozoa must undergo capacitation to fertilize. Capacitation of bovine spermatozoa occurs in vitro in medium supplemented with heparin. Semen cryopreservation is an important tool for assisted reproduction, although the fertility of frozen-thawed spermatozoa is reduced, possibly due to precocious capacitation-like changes that are known to occur. Our purpose was to clarify the mechanisms involved in bull sperm cryocapacitation induced by cryopreservation. Our general hypothesis is that the signaling pathways that lead to capacitation are triggered by the cryopreservation procedure. Ejaculated bovine semen was divided into two aliquots and diluted in extender; one was then kept fresh, whereas the second was cryopreserved. Western blots of extracted sperm proteins with anti-phosphotyrosine antibody showed that capacitation, induced by either heparin in fresh sperm or cryopreservation (cryocapacitation), is associated with a differential profile of phosphotyrosine-containing proteins. Immunolocalization of phosphotyrosine-containing proteins in the fresh and cryopreserved spermatozoa showed that, after thawing, cryocapacitated sperm displayed labeling over the acrosomal region, whereas for fresh sperm, this labeling appeared after 5-h incubation with heparin. The chlortetracycline assay and the ability of the sperm to undergo the lysophosphatidylcholine-induced acrosome reaction were used to confirm that a subpopulation of cryopreserved sperm is capacitated at thawing, irrespective of heparin inclusion. Since glucose is known to inhibit heparin-induced capacitation, the semen extender was modified to include glucose as a means of inhibiting cryocapacitation; however, cryocapacitation was not prevented according to the chlortetracycline assay and profile of phosphotyrosine-containing sperm proteins.

https://bioone.org/journals/Biology-of-Reproduction/volume-69/issue-1/biolreprod.102.011056/A-Differential-Mechanism-Is-Involved-During-Heparin--and-Cryopreservation/10.1095/biolreprod.102.011056.pdf

A Differential Mechanism Is Involved During Heparin- and Cryopreservation-Induced Capacitation of Bovine Spermatozoa

The importance of porcine sperm parameters on fertility in vivo.

Reactive oxygen species-mediated loss of bovine sperm motility in egg yolk Tris extender: protection by pyruvate, metal chelators and bovine liver or oviductal fluid catalase.

Nathaly Cormier

Papers

Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon.

Premature Capacitation of Bovine Spermatozoa Is Initiated by Cryopreservation

A Differential Mechanism Is Involved During Heparin- and Cryopreservation-Induced Capacitation of Bovine Spermatozoa

The importance of porcine sperm parameters on fertility in vivo.

Reactive oxygen species-mediated loss of bovine sperm motility in egg yolk Tris extender: protection by pyruvate, metal chelators and bovine liver or oviductal fluid catalase.