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

A review on goat sperm cryopreservation

Sperm cells are the endpoint of male spermatogenesis and have particular anatomic and metabolic features. Sperm cryopreservation and storage currently require liquid nitrogen or ultralow refrigeration methods for long or short term storage, which requires routine maintenance and extensive space requirements. Conserving sperms have several purposes such as artificial reproductive technologies (ART), species conservation and clinical medicine. The combinations of storage temperature, cooling rate, chemical composition of the extender, cryoprotectant concentration, reactive oxygen species (ROS), seminal plasma composition and hygienic control are the key factors that affect the life-span of spermatozoa. Sperm preservation protocols vary among animal species owing to their inherent particularities that change extenders used for refrigeration and freezing. Extenders for freezing sperm cells contain buffers, carbohydrates (glucose, lactose, raffinose, saccharose and trehalose), salts (sodium citrate, citric acid), egg yolk and antibiotics. The use of different cryoprotectants, like trehalose or glycerol, as well as different concentrations of egg yolk and other constituents in semen extenders are being studied in our laboratory. Several cooling rates have been tested to freeze sperm cells. The use of faster rates (15-60 degrees C/min) gives rise to best sperm survivals after freezing-thawing, but more studies are needed to find the adequate cooling rates for each animal species. Sheep and goat males of some native breeds are being used in studies performed in EZN. Semen from those males has been frozen and stored as part of the Portuguese Animal Germplasm Bank. In small ruminants, individual variations in the quality of frozen semen have been observed, suggesting specific differences in sperm susceptibility to freezing methods, particularly obvious in goat males. Best quality frozen semen from small ruminants is being used in cervical artificial insemination studies aiming to increase productive parameters in selected flocks.

Cryopreservation of domestic animal sperm cells

Mammalian sperm preservation in extenders containing egg yolk (EY) and/or milk has been used for over half a century. However, the mechanism by which EY or milk protects sperm during storage remains elusive. Studies conducted over the past two decades in our laboratory have revealed that a family of lipid-binding proteins (BSP proteins) present in bull seminal plasma is detrimental to sperm preservation since these proteins induce cholesterol and phospholipid removal from the sperm membrane. Interestingly, these detrimental factors of seminal plasma interact with the low-density lipoproteins (LDL) present in EY. This interaction minimizes lipid removal from the sperm membrane, which positively influences sperm storage in liquid or frozen states. Based on several lines of evidence, we suggest that the sequestration of BSP proteins by LDL (BSP proteins: lipoprotein interaction) is the major mechanism of sperm protection by EY. Skimmed milk, which is devoid of lipoproteins, also protects sperm during storage. Several studies indicate that the active components involved in sperm protection by milk are casein micelles. Thus, it appears that the mechanism by which milk protects sperm involves a BSP protein: casein micelle interaction. In view of these new insights, novel strategies have been suggested to improve the efficiency of semen preservation. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc.

New insights towards understanding the mechanisms of sperm protection by egg yolk and milk.

In an attempt to find a suitable freezing method for goat semen, two experiments were conducted to study the influence of trehalose on the cryopreservation of goat spermatozoa. In experiment 1, goat spermatozoa were frozen in trehalose extender (0.375 M) alone (100%) or at different combinations of trehalose with Tris-citric acid-glucose (TCG) extender (0%, 25%, 50%, 75%). Final concentrations of 20% (v:v) egg yolk and 4% (v:v) glycerol were employed in the extenders (osmolality = 370, pH = 7). Sperm motility was assessed using a computer-assisted sperm analysis system and acrosome integrity was assessed using fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA). The sperm-motility parameters improved significantly by increasing the concentration of trehalose (P < 0.05) and significantly high recovery rates for the motility parameters were also achieved by a high concentration of trehalose (P < 0.05). Motility of the frozen-thawed spermatozoa after a 3-h incubation improved significantly with increasing concentrations of trehalose in the extender (P < 0.05). The 75% and 100% trehalose extenders yielded a significant increase in the percentage of spermatozoa with intact acrosome (P < 0.05). In experiment 2, merocyanine 540/Yo-Pro staining was used to study the influence of trehalose on membrane fluidity compared with that of sucrose and TCG. Percentage of cells with high merocyanine fluorescence was significantly higher in spermatozoa treated with trehalose than sucrose or TCG (P < 0.05), indicating a significantly highest membrane fluidity of sperm samples extended with trehalose solution. We thus conclude that the substitution of a Tris-citric acid diluent composition with trehalose significantly improves the freezability of goat spermatozoa. Furthermore, the cryoprotective effects of trehalose observed in this study may be due to enhanced sperm membrane fluidity before freezing.

https://bioone.org/journals/biology-of-reproduction/volume-69/issue-4/biolreprod.103.017889/Trehalose-Enhanced-Fluidity-of-the-Goat-Sperm-Membrane-and-Its/10.1095/biolreprod.103.017889.pdf

Trehalose-Enhanced Fluidity of the Goat Sperm Membrane and Its Protection During Freezing

http://directory.umm.ac.id/Data%20Elmu/jurnal/A/Animal%20Reproduction%20Science/Vol62.Issue1-3.Aug2000/2029.pdf?origin=publication_detail

Production and storage of goat semen for artificial insemination

Chez les races caprines saisonnees, les methodes d'amelioration de production et de conservation de semence doivent prendre en consideration l'influence de l'environnement et en particulier de la photoperiode. Par la fonction qu'elle exerce sur la regulation du developpement gonadique, celle-ci contribue indirectement au controle du comportement sexuel et de la production de sperme, tant du point de vue quantitatif que qualitatif. L'utilisation de traitements photoperiodiques constitue donc un outil efficace pour augmenter la production des doses de sperme destinees a l'insemination artificielle durant la carriere de reproducteur du bouc. Afin d'optimiser la production et la qualite de la semence, une selection prealable des mâles est cependant necessaire. Un probleme specifique dans la conservation de la semence de bouc est l'effet deletere du plasma seminal sur la viabilite des spermatozoides apres congelation / decongelation, principalement lorsqu'ils sont dilues dans des milieux a base de lait ou de jaune d'oeuf. La proteine responsable de cet effet est une enzyme secretee par les glandes bulbo-urethrales appartenant a la famille des lipases pancreatiques et qui possede une double activite lipasique et phospholipasique. La conservation de la semence a l'etat liquide, peu couteuse, est un complement a la cryconservation pour developper l'insemination artificielle a grande echelle. Mais la diminution de la fecondance apres 12 heures de conservation est un frein a l'utilisation de cette technique. Les methodes de congelation disponibles actuellement different sur plusieurs aspects. Les plus efficaces incluent le lavage de la semence pour eliminer le plasma seminal des la collecte.

/pdf/production-et-conservation-de-la-semence-de-bouc-pour-l-o54k3q3xo7.pdf

B. Restall

Papers

Production and storage of goat semen for artificial insemination

Production et conservation de la semence de bouc pour l'insémination artificielle