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result. Glycerol is the most common penetrating cryoprotectant for sperm and enters the cell via passive diffusion. Glycerol also causes injury directly to cells, including protein denaturation and alteration of the plasma membrane (Macedo et al., 2018 ; Moore et al., 2006 ; Vafaei et al., 2019 ). The search for a freezing medium which in- cites minimal osmotic damage on the sperm cell is ongoing. Amides (methyl formamide or dimethyl formamide) are another category of penetrating cryoprotectants. Amides are of lower molecular weight than glycerol, allowing faster diffusion and less osmotic damage (Alvarenga et al., 2005 ; Macedo et al., 2018 ; Squires et al., 2004 ). Commercial freezing extenders generally contain a combination of glycerol and an amide, as well as non- penetrating cryoprotectants (Moffett et al., 2016 ; Sieme, 2011b ). These extenders also help mi- nimise the formation of ice crystals within the sperm cell (Moore et al., 2006 ; Watson, 2000 ). During cryopreservation, the cell is left with unfrozen hyperosmotic channels after the loss of water from the cell. If cellular dehydration and cell shrinking happen too quickly as a result of a too- fast cooling rate, water freezes inside the cell membrane before it has a chance to move extracellularly (Loomis & Graham, 2008 ). During semen centrifugation prior to cryopreservation, a vari-
able amount of seminal plasma is removed with the supernatant. The majority of the fluid component of the stallion ejaculate is composed of seminal plasma contributed by the accessory sex glands. Studies have described the importance of retaining 5%–20% seminal plasma in the ejaculate post- centrifugation in order to increase cryosur- vival (Sieme, 2011a ). In some cases, the seminal plasma from a given stallion has been shown to improve the fertility of cryopreserved semen. Multiple investigations have identified a stallion- specific ef- fect of seminal plasma on the fertility of cryopreserved semen, but there is no consensus regarding this effect (Aurich et al., 1996 ; Katila et al., 2002 ; Moore et al., 2005 ). Some ‘poor freezers’ can benefit from the addition of seminal plasma from a ‘good freezer’ (Arruda et al., 2008 ; Moore et al., 2005 ; Neuhauser et al., 2019 ). The ad- dition or removal of seminal plasma to stallion semen intended for cryopreservation is an area of potential manipulation to improve cryosurvival.
THAWING AND HANDLING SEMEN
Cryopreserved semen can be stored indefinitely in a liquid nitro- gen tank maintained at −196°C. Straws are loaded into goblets (4–5 straws per goblet) and goblets are then loaded onto canes (2 goblets per cane). In addition to identification listed on each indi- vidual straw, the top of each cane should be labelled with the stal- lion ' s name and freeze date. A database of cryopreserved semen should be maintained and kept up to date as semen is shipped or used. Prior to inseminating a mare, a Styrofoam box is filled with 3–6 cm of liquid nitrogen. The cane containing the desired semen is moved quickly from the tank to the Styrofoam box, minimising exposure to room temperature air. The desired number of straws are removed from the goblets and canes while remaining beneath
PASCH
the liquid nitrogen surface, and the straws are read to confirm the appropriate semen will be thawed. Depending on the freez- ing method and extender used, 0.5 mL straws are thawed at either 37°C for 30 s or 46°C for 20 s in a pre- warmed water bath. The straws are removed promptly and dried thoroughly, then flicked or shaken to move the air bubble to the sealed (i.e., non- cotton plug) end of the straw.
MARE MANAGEMENT
The mare aspect of the breeding process is tightly managed to op- timise the time and location of frozen–thawed semen deposition. Mares are ideally bred within 6 h of ovulation using a deep horn artificial insemination technique which allows for the placement of semen directly adjacent to the oviductal papilla ipsilateral to the ovulation. It is beyond the scope of this article to delve into the nu- ances of mare management for frozen semen breeding, but an over- view of the process follows. Mares are examined in early oestrus, allowing time for pre- breeding diagnostics as warranted. When the mare is in oestrus with a minimum 35 mm follicle, an ovulatory agent such as deslorelin acetate (SucroMate, ThornBioscience) is adminis- tered to induce ovulation in approximately 40 h. Mares are examined frequently for ovulation, typically every 6 h as expected ovulation approaches. Upon the detection of ovulation, straws are thawed as described above then loaded one at a time into a specially designed insemination pipette and semen is advanced with a stylet designed for that purpose. Semen is deposited at the uterotubal junction ip- silateral to the side of ovulation. Each straw is removed before the next straw is advanced. Expected per cycle pregnancy rates with fro- zen semen range from 40% to 60% (Metcalf, 2007 ), although these can be highly variable given the multifactorial nature of breeding. Deep horn insemination (as opposed to uterine body insemination) has been shown to improve pregnancy rates with relatively low- dose frozen semen breeding (Brinsko et al., 2003 ; Buchanan et al., 2000 ; Govaere et al., 2014 ); no minimum threshold of sperm number has been established.
POST- THAW ASSESSMENT OF SPERM
As alluded to above, there are no standardised parameters for frozen stallion semen doses. General industry guidelines have historically suggested a minimum of 250 million progressively motile morpho- logically normal sperm per dose (Metcalf, 2007 ). The once ‘standard’ dose of 8 × 0.5 mL straws has given way, at times, to as few as 1–2 straws per dose, based on evidence of pregnancies being achieved at ever- decreasing semen volumes and sperm numbers (Buchanan et al., 2000 ; Govaere et al., 2014 ; Hayden et al., 2012 ). This trend is further propagated by the popularity of stallions who may be de- ceased, aged or otherwise unable to continually produce semen for cryopreservation, in an effort to ‘conserve’ semen. Given the often low numbers of straws, there is rarely enough semen remaining in a
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