AN OVERVIEW OF CRYOPRESERVATION AND CAUSES OF SPERM DAMAGE
breeding dose to warrant complete semen analysis. Subjective mo- tility assessment may provide a comfort to the clinician that live and motile sperm have been inseminated. Using a small drop remaining in the insemination pipette, subjective post- thaw total and progres- sive motility can be observed under the microscope on a warmed slide with a cover slip. This sample may not be representative of the entire dose due to its small volume and potential contamination with mucus from the mare ' s reproductive tract. A thorough and more in- formative post- thaw semen analysis would include objective total and progressive motility assessment, membrane- intactness assay, morphologic assessment, acrosome- intactness assay and confor- mation of total sperm number per straw, while acknowledging that the truest test of fertility is a breeding trial. Although in vitro sperm quality and fertility are related, there is no direct linear relationship (Love, 2018 ).
EPIDIDYMAL SPERM CRYOP RES ERV ATION
In the event of untimely death or elective castration, epididymal sperm can be recovered, processed and cryopreserved with result- ing pregnancies. Sperm transport through the epididymis allows for maturation and changes in the sperm plasma membrane in response to epididymal secretions, rendering sperm recovered from the distal- most portion of the epididymis (the cauda epididymis or epididy- mal tail) capable of fertilisation (Sostaric et al., 2008 ). A number of publications offer instructions to collect and process epididymal sperm (Bruemmer, 2006 ; Monteiro et al., 2011 ; Papa et al., 2008 ). Guidelines have been developed for shipping epididymides at 5°C for sperm harvesting and processing 24–48 h after death or castra- tion (Stawicki et al., 2016 ), and others have examined the addition of seminal plasma to epididymal sperm (Moore et al., 2005 ; Morris et al., 2002 ).
FUTURE DIRECTIONS
Artificial insemination with cryopreserved- thawed equine semen remains challenging, due to its relatively low success rate (compared with cooled or fresh semen) and its necessitation of intensive mare management. A number of newer investigations aim to improve the convenience and accessibility of frozen semen. A group from Colorado (Prell et al., 2018 ) investigated the process of thawing fro- zen semen, extending and storing at 5°C for 24 h before insemina- tion, and found that their protocol resulted in acceptable pregnancy rates. Several groups have looked at ways to facilitate shipping cooled semen to a suitable facility for cryopreservation (Ferrer et al., 2020 ; Melo et al., 2007 ). In these situations, semen is collected and extended at one facility, then cooled and shipped overnight at 5°C to a second facility. The intricacies of these studies are beyond the scope of this paper but demonstrate that stallion semen can be cooled and shipped prior to freezing with acceptable post- thaw pa- rameters and pregnancy rates.
ETHICS STATEMENT Not applicable to this review article.
ORCID Lauren Pasch
https://orcid.org/0000-0003-2653-8178
REFERENCES
Alvarenga , M.A. , Papa , F.O. , Landim- Alvarenga , F.C. & Medeiros , A.S.L. ( 2005 ) Amides as cryoprotectants for freezing stallion semen: a re- view . Animal Reproduction Science , 89 , 105 – 113 .
Alvarenga , M.A. , Papa , F.O. & Neto , C.R. ( 2016 ) Advances in stallion semen cryopreservation . The Veterinary Clinics of North America. Equine Practice , 32 , 521 – 530 .
Arruda , R.P. , Andrade , A.F.C. , Raphael , C.F. , Peres , K.R. , Nascimento , J. , Martins , S.M. et al. ( 2008 ) Effects of addition of seminal plasma on lifespan of frozen- thawed equine spermatozoa . Animal Reproduction Science , 107 ( S3–4 ), 307 – 308 .
Aurich , J.E. , Kuhne , A. , Hoppe , H. & Aurich , C. ( 1996 ) Seminal plasma af- fects membrane integrity and motility of equine spermatozoa after cryopreservation . Theriogenology , 46 , 791 – 797 .
Barbacini , S. , Marchi , V. & Zavaglia , G. ( 1999 ) Equine frozen semen: re- sults obtained in Italy during 1994–1997 period . Equine Veterinary Education , 11 , 109 – 112 .
Brinsko , S.P. , Rigby , S.L. , Lindsey , A.C. , Blanchard , T.L. , Love , C.C. & Varner , D.D. ( 2003 ) Pregnancy rates in mares following hystero- scopic or transrectally- guided insemination with low sperm num- bers at the utero- tubal papilla . Theriogenology , 59 , 1001 – 1009 .
Bruemmer , J.E. ( 2006 ) Collection and freezing of epididymal stallion sperm . The Veterinary Clinics of North America. Equine Practice , 22 , 677 – 682 .
Buchanan , B. , Seidel , G. , McCue , P. , Schenk , J. , Herickhoff , L. & Squires , E. ( 2000 ) Insemination of mares with low numbers of either un- sexed or sexed spermatozoa . Theriogenology , 53 , 1333 – 1344 .
Clulow , J.R. , Mansfield , L.J. , Morris , L.H.A. , Evans , G. & Maxwell , W.M.C. ( 2008 ) A comparison between freezing methods for the cryopres- ervation of stallion spermatozoa . Animal Reproduction Science , 108 , 298 – 308 .
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CONCLUSION
The semen cryopreservation process causes multiple insults result- ing in overall decreased fertility of cryopreserved- thawed semen. Despite this, artificial insemination of mares with cryopreserved- thawed semen remains popular and even required (due to stallion availability) in some breeds. Cryopreservation leads to sperm loss and irreversible sperm damage, which hinders pregnancy rates. Precise and intensive mare management, along with an increased understanding of the factors contributing to cryosurvival, help mod- ulate the freezing- induced fertility decline.
CONFLICT OF INTEREST STATEMENT No conflicts of interest have been declared.
FUNDING INFORMATION None.
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