6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518496

Effects of single layer density gradient centrifugation oncooled-stored stallion semen

R.R.D. Maziero, P.M. Papa, F.P. Hartwig, F.P. Lisboa,J.A. Delláqua, Jr., M.A. Alvarenga, P.N. Guasti,F.C. Landim-Alvarenga, and F.O. PapaDepartment of Animal Reproduction and VeterinaryRadiology, School of Veterinary Medicine and AnimalScience, UNESP, Botucatu, SP18610-970, Brazil

The objective of this studywas to evaluate effects of a singlelayer density gradient EquiPure (Nidacom) centrifugationon cooled-stored stallion semen. Each of two ejaculatescollected from 10 stallions was divided into three aliquots:control, centrifuged and treatment. In the control group,semen was diluted with a skim milk-based extender(Botu-Sêmen) at a concentration of 50x106sperm/ml. Incentrifuged and treatment groups, samples with 1x109

spermatozoa were diluted with Botu-Semen 1:1 (v:v) andcentrifuged at 600xg for 10 minutes. The supernatant of thecentrifuged groupwas removed and the pellet resuspendedin Botu-Semen to a final concentration of 50x106sperm/mL.In the treatment group, the pellet was resuspended in 5 mlof Botu-Semen. This sample was placed upon 5 ml ofdensity gradient in a 15 ml tube and centrifuged at 400xgfor 20 minutes. The pellet was recovered and diluted inBotu-Sêmen to a final concentration of 50x106 sperm/ml.All samples were cooled in an isothermal box (Botu-Flex) at5 oC for 24 hours. Semen was evaluated before and aftercooling by CASA for total motility (TM), progressivemotility(PM), rapid sperm (RAP); plasmamembrane integrity (PMI)was evaluated by epifluorescence microscopy. All parame-ters were analyzed by ANOVA, followed by Tukey's test toidentify significant differences (P < 0.05).The average recovery rate after centrifugation with thedensity gradient was 41%. Before cooling, the treatmentgroup was superior in all parameters when compared tocentrifuged samples, and demonstrated higher values of PMand RAP compared to control. However, control groupshowedhigher valuesof TMandPMI than centrifugedgroup.After cooling, treatment group was greater compared tocontrol and centrifuged groups in all parameters evaluated(P < 0.05). In conclusion, the centrifugation with a densitygradient was effective in selecting sperm more resistant tocooling at 5oC for 24 hours. This gradient may be an impor-tant option to improve the fertility of “bad cooler” stallions.

Acknowledgments

FAPESP/Botupharma�.

Table 1Mean values for total motility (TM), progressivemotility (PM), percentage of rapidpost-cooling semen samples for control, centrifuged and EquiPure groups (stora

Pre-cooling

Control Centrifuged Treatmen

TM (%) 76a 71b 81a

PM (%) 31b 29b 43a

RAP (%) 66b 60b 73a

PMI (%) 62a 55b 65a

a,b Different letters in a row indicate differences in pre-cooling parameters (P <A,BDifferent letters in a row indicate differences in post-cooling parameters (P <

Fertility of equine epididymal frozen semen usingsperm motility stimulants

C.M. Melo-Oña 1, L.C.O. Magalhães 1, F.O. Papa 1,I. Martin 1, F.C. Landim-Alvarenga 1, J.N.P.P. Filho 2,R.F. Soares 1, M.A. Alvarenga 1, G.A. Monteiro 1, andJ.A. Dell'Aqua, Jr. 11 DRARV, Botucatu-SP, Brazil, 2 D.P.A. São Paulo StateUniversity, Botucatu-SP, Brazil

Epididymal sperm has a poor motility, probably due to lackof exposure to activating factors from seminal plasma.Effects of seminal plasma and sperm motility factors suchas caffeine, progesterone, heparin and PHE (penicillamine,hypotaurine and epinephrine) have been investigated.Nevertheless, the influence of these substances on fertilityremains unclear, despite an increase in epididymal spermmotility. The aim of the present study was to evaluate theeffect of sperm motility factors (caffeine, heparin, PHE) onfertility of mares inseminated with epididymal sperm.Equine epididymal samples were obtained from a pool of10 stallions using retrograde flush with 40mL of Botu-Semen per epididymal cauda [1]. The samples were dividedinto 3 groups: Botu-Semen (BS, control), Fert-Talp andSperm-Talp, diluted 1:1 with these media containing100x106 spermatozoa per straw, and then incubated atroom temperature (25oC) for 15 min. The samples werecentrifuged (1000 x g for 10 min), the supernatant wasremoved and the pellets were resuspended in Botu-Crio.Samples were loaded in 0.5mL straws and cooled at 5 �C for20 min. Then they were frozen in nitrogen vapor (6 cmabove the level of liquid nitrogen) for 20 min and finallyplunged into nitrogen and stored. Straws were thawed at46 �C for 20 s. Thirty mares were used for the fertility trial.Ovulation was induced with 1 mg (i.m.) of deslorelinacetatewhen the follicle achieved 35–40mmdiameter. Thefirst insemination was done 36 h after ovulation inductionwith 400�106 sperm cells and the second AI with the samesperm amount 6 h after ovulation. Inseminations wereperformed in the tip of the uterine horn with 10 insemi-nations per group. The samples were analyzed by CASA andplasma membrane integrity was evaluated by carboxy-fluorescein diacetate and propidium iodide staining. Datawere analyzed by ANOVA followed by Tukey test (P< 0.05).Fertility rates were evaluated by Chi-square test. Nodifferences in fertility were observed among groups.Pregnancy rates were 80%, 60% and 80% for samples usingBS (control), Fert-Talp and Sperm-Talp, respectively. Totalmotility (43.0 � 20.75a; 69.0 � 14.18b and 70.5 � 12.83b),progressive motility (14.0 � 11.97a; 31.0 � 8.87ab and

sperm (RAP) and plasmamembrane integrity (PMI) in the pre-cooling andge at 5oC for 24 hours)

Post-cooling

t Control Centrifuged Treatment

46B 49B 70A

19B 18B 34A

35B 34B 55A

52B 49B 63A

0.05).0.05).