Maintenance of low temperatures is the key to successful storage of frozen semen. During cooling and freezing, microenvironments are created within the semen straw. Each chemical component of extended semen freezes or solidifies at a different temperature. Water begins to freeze as temperatures are decreased below 0 °C, forming ice crystals that remain somewhat unstable at temperatures above –80 °C.³ This instability is thought to be caused by recrystallization of the ice. Also, as water is converted to ice, sperm are exposed to the remaining concentrated solution of salts and other components of the extender which freeze at temperatures considerably below the freezing point of water. Instability of ice and concentrated solutions are harmful to sperm. Dehydration during cryopreservation has an important impact on sperm; dehydration decreases the risk of intracellular ice crystal formation, but excessive dehydration is also detrimental.^2,4 Adjusting the osmolality of the diluents is critical, as it influences water fluxes during cryopreservation.^2,4 Fortunately, cryoprotective agents and optimized freezing programs help minimize sperm damage. However, semen must be kept well below critical temperatures where the recrystallization of ice begins to occur (–100 to –80 °C). Temperatures fluctuate dangerously from the low to upper third of the neck of the tank³ (Table 32.1). Furthermore, stored semen can be exposed to adverse high temperatures when removed from the tank for thawing, when transferring semen from tank to tank, and when handling semen within the neck when trying to locate and thaw a specific straw. In addition, other semen straws in goblets and canisters are also exposed to high temperatures. The thermal response of semen in 0.5-mL straws when raised to 5 cm from top of the tank (exposed to temperature of –22 °C) and 2.5 cm (exposed to 5 °C) is shown in Table 32.2.³ The time to reach critical ice recrystallization temperature (–100 to –80 °C) is approximately 10–20 s for both temperatures. Thermal injury to sperm is permanent and cannot be corrected by returning semen to liquid nitrogen. To assure maintenance of sperm viability, canes and canisters should be raised into the neck of the tank and kept below the frost line only for 5–8 s. If necessary, the canister should be lowered back into the tank for cooling, and subsequently brought back to the neck of the tank.

The number of sperm per straw is variable, with some reduction in number common for high-demand elite bulls, and substantially lower numbers for sex-sorted semen. Higher dilutions (2 million sperm per 0.25-mL straw) did not affect the proportions of linearly motile spermatozoa, membrane integrity or stability, nor chromatin integrity immediately after thawing compared with a low dilution rate (15 million sperm per 0.25-mL straw). Further, there was no difference in pregnancy rate between dilution rates.⁵ Regardless, it is well known that some bulls require more sperm per insemination dose to maintain fertility. Furthermore, due to very low numbers per insemination dose, the fertility of sex-sorted sperm is almost inevitably decreased.

Semen thawing method

Various methods for thawing semen in straws have been recommended, including a variety of water bath temperatures and thawing periods, shirt pocket thawing, air-thawing, and thawing in the cow.⁶ Regardless, it is recommended that all persons on a farm use the same method to thaw semen. The National Association of Animal Breeders recommends a semen straw be immersed in a water bath at 30–35 °C for 40 s. The thawing time for straws should be a minimum of 30 s.⁷ Thawed semen should be inseminated within 15 min after thawing and drastic decreases in post-thaw temperature must be avoided.⁸

In pocket thaw methods, the initial pocket temperature was 30 ± 1 °C. However, 1 min after pocketing, the temperature decreased to approximately 19 ± 1 °C, which could have adverse effects on sperm parameters. Semen in straws began to liquefy approximately 3 min after pocketing, with thawing completed by 5 min.⁸ The results of semen viability assays after pocket thaw differed widely among experiments. The rate of thawing in a shirt pocket could be quite variable depending on the ambient temperature and the insulating effect of different types and thicknesses of clothing. Furthermore, the thaw rate was much slower for pocket-thaw compared with a water bath.⁸ More rapid thaw rates result in higher post-thaw viability compared with the pocket-thaw method.⁸ However, a recent study claimed that irrespective of improved in vitro semen quality with a fast thaw rate, these semen quality measures did not increase in vivo fertility compared with a pocket-thaw method.⁹ If frozen semen is prepared to permit flexible thawing, the thaw method used, whether pocket or warm water thaw, should not affect conception under commercial conditions.⁹ Although some sire and extender (egg-yolk citrate vs. nonheated whole milk extender) combinations seem to be tolerant to thaw procedures, other combinations are more sensitive, resulting in reduced post-thaw sperm survival, conception rates, or both, in response to thaw methods.¹⁰

Concurrently thawing multiple straws of semen can facilitate efficiently breeding a large number of cows without compromising semen quality or conception rates. Despite several reports that more than one straw can be safely thawed, caution must be employed while preparing multiple guns, because it is paramount to follow proper procedures for straw preparation and thermal protection and to always work within skill and time constraints. An experienced inseminator can thaw multiple straws of semen and prepare insemination guns to breed up to four cows within 20 min, without an adverse effect on conception.¹¹ Another study concluded that, on average, conception rates differed between professional AI technicians and herd inseminators. However, elapsed time from initial thaw to completion of fourth AI and sequence of insemination (first, second, third, or fourth) had no effect on conception rate within inseminator group.¹² Oliveira et al.¹³ concluded that sequence of insemination after simultaneous thawing of 10 semen straws can differently affect conception rates at timed AI, depending on the sire used. Regardless of number of straws thawed simultaneously, it is vital that straws are agitated immediately after immersion to prevent straws from clumping together, resulting in a decreased rate of thaw.

Semen handling after thawing

Mixing water and semen will cause irreversible sperm injury. Hence, on removal of the straw from the water bath, the straw should be dried (typically with a paper towel) before it is cut. If the straw is defective (e.g., perforated), it should be discarded.

Prevention of cold shock is critical for post-thaw semen handling. Cold shock inflicts irreversible injury to sperm and is caused by rapid post-thaw decrease in temperature; this occurs when semen is thawed and then subjected to cold environmental temperatures before being inseminated. Cold shock decreases motility and fertilizing competency due to irreversible changes in the sperm plasma membrane. The severity depends on the rate and duration of temperature drop. The most obvious sign of cold shock is loss of motility which is not regained on warming the semen. There is also a decrease in the rate of fructose breakdown by the sperm and a decrease in oxygen uptake and in ATP concentrations, which can now be no longer synthesized and used to supply energy. Cold shock caused an influx of sodium (186.2 ± 9.3 vs. 140.7 ± 10.2 mg/100 g wet weight) into sperm, as well as an efflux of potassium (101.6 ± 6.8 vs. 155.4 ± 9.6 mg/100 g wet weight) and magnesium (8.1 ± 0.2 vs. 11.8 ± 0.5 mg/100 g wet weight) from the sperm.¹⁴ The higher total calcium concentrations in bovine seminal plasma than in sperm are primarily due to a much higher concentration of complex calcium in seminal plasma. The concentration of protein-bound calcium was approximately the same in spermatozoa and seminal plasma, whereas concentrations of ionized calcium were lower in seminal plasma than in spermatozoa. Cold shock significantly increased the concentration of total calcium in spermatozoa, with a corresponding decrease of total calcium in the seminal plasma, due to a decreased content of ionized calcium in the seminal plasma and a simultaneously increased content of complex and protein-bound calcium in spermatozoa. In contrast, there was no increase of calcium in spermatozoa following slow cooling.

Cold shock occurs most frequently when breeding is performed in cold weather and especially when a warm water bath is used. The high surface to volume ratio of a straw makes it vulnerable to cold shock. Saacke,¹⁵ using the 0.5-mL French straw, measured the effect of static ambient temperatures (21, 4 and 16 °C) on the rate of temperature drop in semen after thawing during preparation of the inseminating rod (Table 32.3). The temperature drop was only 3–6 °C for an ambient temperature of 21 °C; furthermore, warming the rod was effective in countering temperature drop. However, during preparation at 4 and 16 °C ambient temperatures, the drops were 15 and 20 °C, respectively, and preparation of the AI gun for insemination at these two temperatures only postponed the temperature drop. Clearly, precautions against cold shock must be implemented during preparation of inseminating guns in a cool environment.