E-Z Freezin® CryoMax™ Freezing Extenders  

CryoMaxTM LE and CryoMaxTM MFR5 are new Equine Freezing Extenders that were developed by Animal Reproduction Systems in conjunction with Colorado State University.  Initial testing for these extenders were performed at the Colorado State University Equine Reproduction Laboratory. During the 2015 breeding season, the majority of their client stallion semen was cryopreserved using one of the new CryoMaxTM freezing extenders.

Preliminary data showed that overall, the new CryoMaxTM Equine Semen Freezing Extenders improved post-thaw total and progressive motility by an average of 10 % over other commercially available extenders.  In addition, the new CryoMaxTM extenders yielded a markedly higher post-thaw motility in five stallions with low post-thaw progressive motility (< 35 %) when frozen in their original extenders. Post-thaw progressive motility in the poor-freezer stallions improved from 30 % to 58 %, for an average improvement of 28 %.

.
a.
Stallion 1.  Post-thaw motility in original extender was 22 %; post-thaw motility in new CryoMax™ LE was 53 %
(31 % improvement).
b.
Stallion 2.  Post-thaw motility in original extender was 31 %; post-thaw motility in new CryoMax™ LE was 73 %
(42 % improvement).
c.
Stallion 3.  Post-thaw motility in original extender was 34 %; post-thaw motility in new CryoMax™ MFR5 was 56 %
(22 % improvement).
d.
Stallion 4. Post-thaw motility in original extender was 31 %; post-thaw motility in new CryoMax™ LE was 52 %
(21 % improvement).
e.
Stallion 5. Post-thaw motility in original extender was 32 %; post-thaw motility in new CryoMax™ MFR5 was 57 %
(25 % improvement).
It should be noted that post-thaw motility and potential for fertility with frozen thawed semen are not necessarily synonymous. Most of the semen frozen with the new CryoMaxTM extenders is destined to be used in future breedings.  So far this past season, four mares were bred using semen frozen in CryoMaxTM extenders and three became pregnant on their first cycle.

Overall, the new CryoMaxTM series of equine semen freezing extenders demonstrated superior post-thaw sperm motility in 73 % of stallions (8 out of 11) as compared to other commercially available extenders.
A series of studies were performed at the Equine Reproduction Laboratory, Colorado State
University to compare commercial extenders for cryopreservation of stallion semen.  Three
freezing extenders contained a combination of glycerol and an amide as cryoprotectants
(BotuCrio®, CryoMaxTM LE and CryoMaxTM MFR5), while two freezing extenders utilized
glycerol as the only cryoprotectant (LE, MFR5).

Study 1.  Semen was collected from a total of 12 different stallions.  Aliquots of each ejaculate
were diluted in one of five different extenders, loaded into 0.5 ml straws, cooled to 5 to 8° C
over a 30 to 60 minute period and subsequently cryopreserved. One representative straw from
each extender for every stallion was thawed in a 37° C water bath for 30 seconds.  The thawed
sperm was allowed to warm for 10 minutes and then evaluated for total and progressive
motility using a computer assisted semen analysis (CASA) system.  The highest average post-
thaw motility was noted for the CryoMaxTM LE extender (Table 1).
Table 1.  Post-thaw motility for spermatozoa from 12 stallions cryopreserved in each of 5
commercial freezing extenders. Data are presented as the mean ± SEM.
Extender Total Motility (%) Progressive Motility (%)
BotuCrio® 50.4 ± 4.6a,x 39.8 ± 4.3a
CryoMaxTM LE 57.4 ± 4.4a,y 43.5 ± 4.6a
CryoMaxTM MFR5 51.7 ± 4.2a 38.7 ± 4.4ab
LE 38.3 ± 4.1b 27.5 ± 3.8b
MFR5 36.4 ± 4.4b 25.7 ± 4.0b
ab Values within a column with different superscripts are significantly different (p<0.05) [ANOVA]
xy Values are statistically different (p=0.032) [paired T-test]

Study 2.  Semen was collected from 30 different stallions.  Aliquots of each ejaculate were diluted in one of four commercial extenders, loaded into 0.5 ml straws, cooled to 5 to 8° C and cryopreserved. One straw from each extender for every stallion was thawed in a 37° C water bath for 30 seconds.  The thawed sperm was allowed to warm for 10 minutes and then evaluated for total and progressive motility using a CASA system.  Again, the highest average post-thaw motility was noted for the CryoMaxTM LE and CryoMaxTM MFR5 extenders (Table 2).

Table 2.  Post-thaw motility for spermatozoa from 30 stallions cryopreserved in each of 4 commercial freezing extenders (mean ± SEM).
Extender Total Motility (%) Progressive Motility (%)
CryoMaxTM LE 60.2 ± 2.7a 47.5 ± 2.9a
CryoMaxTM MFR5 55.0 ± 2.3a 43.4 ± 2.4a
LE 42.5 ± 4.0b 31.5 ± 3.7b
MFR5 39.0 ± 3.5b 29.1 ± 3.2b
Summary. Freezing extenders containing a combination of glycerol and amide cryoprotectants yielded better post-thaw sperm motility than extenders containing glycerol alone.  CryoMaxTM LE extender yielded a higher post-thaw total motility when directly compared to BotuCrio® extender.
Studies from 2015 Season
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