Advances in cryopreservation of stallion semen in modified INRA82.
In the procedure used in this paper, semen was first diluted in INRA82+2% egg yolk (E1) at 37 degrees C. Before or after cooling to 4 degrees C, semen was centrifuged and diluted in E1+2.5% glycerol (E2). Cooled semen was frozen in 0.5-ml straws. Straws were thawed at 37 degrees C for 30s. For fertility trials, frozen ejaculates were used only if total post-thaw motility was above 35%. Most mares were inseminated two times before ovulation with 400 x 10(6) total spermatozoa every 24h. This paper presents post-thaw motility (CASA) and fertility results obtained when some steps of the procedure were evaluated. Use of the first three jets of ejaculate before the centrifugation did not improve post-thaw motility compared to use of the whole semen (25% versus 25%, 2 stallions x 12 ejaculates, P>0.80). When the first dilution was performed in E2 at 22 degrees C instead of in E1 at 37 degrees C, motility was slightly improved (38% versus 36%, n>283 ejaculates per group, P<0.04) but fertility was similar (51% versus 58%, n>196 cycles per group, P>0.10). Coating the spermatozoa with 0.5, 1, 2, 4 and 8mM of Concanavalin A resulted in unchanged post-thaw motility (6 stallions x 3 ejaculates, P>0.05). The extender E2 was modified or supplemented with different substances. Increasing egg yolk concentration from 2 to 4% (v/v) did not increase post-thaw motility (42% versus 34%, 6 stallions x 2 ejaculates, P>0.05). Different glycerol concentrations (range: 1.7-3.7%) had no significant effect on post-thaw motility even though 2.4-2.8% resulted in a nonsignificant higher motility (7 stallions x 2 ejaculates, P>0.05). Glutamine at 50mM in E2 improved post-thaw motility compared with no glutamine (49% versus 46%, n>584 ejaculates per group, P<0.0001) but not fertility (53% versus 54%, n>451 cycles per group, P>0.80). Thawing at 75 degrees C for 10s slightly increased motility after 120 min at 37 degrees C (6 stallions x 1 ejaculate, P<0.05) but no effect on per-cycle fertility was noted (32% (19 cycles) versus 41% (17 cycles), P>0.50). When post-thaw dilution was performed using a fixed molarity multi-step system (25 mOsm per step) from various osmolarities (900-690 mOsm) to 365 mOsm, motility was unaffected compared with dilution in one step (36% versus 38%, 6 stallions x 1 ejaculate, P>0.20).
Vidament
M
M
Reproduction Equine, Haras Nationaux/INRA, PRC, 37380 Nouzilly, France. vidament@tours.inra.fr
Yvon
J M
JM
Couty
I
I
Arnaud
G
G
Nguekam-Feugang
J
J
Noue
P
P
Cottron
S
S
Le Tellier
A
A
Noel
F
F
Palmer
E
E
Magistrini
M
M
D000818:Animals; D003208:Concanavalin A / Q000494:pharmacology; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003451:Cryoprotective Agents; D004530:Egg Yolk / Q000502:physiology; D005260:Female; D005973:Glutamine / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D006736:Horses / Q000502:physiology*; D008297:Male; D011247:Pregnancy; D012661:Semen; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*
D003451:Cryoprotective Agents; D005973:Glutamine; D003208:Concanavalin A; D005990:Glycerol