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Improvement of cooled equine semen by addition of carnitines
Journal of Equine Veterinary Science 34 (2014) 48
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Improvement of cooled equine semen by addition of carnitines F.L. Lisboa 1, *, F.P. Hartwig 1, C.P. Freitas-Dell’Aqua 1, F.P. Hartwig 2, F.O. Papa 1, J.A. Dell’aqua Jr. 1 1 2
Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, UNESP, Botucatu, SP 18610-970, Brazil Department of Social Medicine, College of Medicine, UFPel, Pelotas, Brazil
acrosomal integrity were determined by Hoeschst 33342, propidium iodide and FITC-PSA, generation of reactive oxygen species (ROS) by carboxy-H2DCFDA, membrane lipid peroxidation by C11Bodipy and DNA fragmentation by acridine orange staining. Samples were analyzed using flow cytometry (BD LSR Fortessa; Becton Dickinson, Mountain View). Evaluations were performed in fresh semen (0H), 24 hr (24H) and 48 hr (48H) of cooling. A permutation-based ANOVA with repeated measures was used to assess the effect of the treatment on each sperm parameter at each incubation time. When p<0.05, the post-hoc analysis was performed by pairwise paired t-tests with Holm’s correction. After 24 hr of storage, LC yielded significantly higher percentages of TM and PM than control, while LC/AC showed better PM. At 48 hr, treated groups showed significantly better motility parameters than control, except AC, where VSL was similar to control and LC/AC. Although no differences were found in flow cytometric parameters, it was noted that treated groups were numerically greater then control in some parameters, and probably with a larger number of ejaculates this numerical trend can be
L-carnitine (LC) and acetyl-L-carnitine (AC) modulate several sperm metabolic functions, such as fatty acid oxidation, acetyl-CoA/free CoA ratio and utilization of pyruvate and lactate as energy substrates. LC has a powerful antioxidant effect by reducing the availability of lipids to peroxidation and increasing activity of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase. Moreover, the presence of carnitines ensures the operation of oxidative pathways by reducing acetylCoA levels and provides acetyl groups for sperm motility. The aim of this study was to evaluate the effect of these substances on cooled sperm viability. Two ejaculates from 6 stallions were diluted with skim milk-based extender (Botu-SemenTM) to a final concentration of 50x106 spermatozoa/mL and then divided into 4 groups: Control (no LC/AC), LC (0.1mM/mL of LC), AC (0.1mM/mL of AC) and LC/ AC (0.1mM/mL of LC+0.1mM/mL of AC). All groups had the osmolarity adjusted to 380 mOsm and pH at 6.8. After treatment, samples were placed at 5 C for 24 and 48 hr. Sperm motility was determined by computer-assisted semen analysis (Hamilton-ThorneTM) for total motility Table 1. Mean values of TM, PM, VSL and RAP analyzed at 0, 24 and 48 hr.
TM PM VSL RAP a,b
0H
24H
C
C
LC
AC
LC/AC
C
48H LC
AC
LC/AC
68.912.7 33,810.2 94,710.7 62.114.0
50.316.2a 2512.6a 86.89.5 41.414.3
59.112.1b 3314.9b 92.614.5 49.014.4
53.417.6a 29.814.0a 89.810.6 45.516.7
56.814.6a 33.312.8b 91.811.5 47.512.1
32.216.5a 12.19.7a 72.113.7a 23.715.6a
45.017.5b 22.814.3b 84.616.3b 35.417.5b
40.316.7b 1913.2c 77.615.3a,c 30.116.9b
43.717.6b 21.613.5b,c 82.215.4b,c 33.416.8b
Values in the same row with different superscript differ significantly (p<0.05).
(TM), progressive motility (PM), average path velocity (VAP), straight line velocity (VSL), curvilinear path velocity (VCL) and rapid sperm (RAP). Plasma membrane and
* Presenting author E-mail address: [email protected] (F.L. Lisboa).
confirmed. Based on these results, it was concluded that the addition of L-carnitine and Acetyl-L-Carnitine improves the maintenance of sperm motility after 24 hr storage at 5 C.
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Improvement of cooled equine semen by addition of carnitines F.L. Lisboa 1, *, F.P. Hartwig 1, C.P. Freitas-Dell’Aqua 1, F.P. Hartwig 2, F.O. Papa 1, J.A. Dell’aqua Jr. 1 1 2
Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, UNESP, Botucatu, SP 18610-970, Brazil Department of Social Medicine, College of Medicine, UFPel, Pelotas, Brazil
acrosomal integrity were determined by Hoeschst 33342, propidium iodide and FITC-PSA, generation of reactive oxygen species (ROS) by carboxy-H2DCFDA, membrane lipid peroxidation by C11Bodipy and DNA fragmentation by acridine orange staining. Samples were analyzed using flow cytometry (BD LSR Fortessa; Becton Dickinson, Mountain View). Evaluations were performed in fresh semen (0H), 24 hr (24H) and 48 hr (48H) of cooling. A permutation-based ANOVA with repeated measures was used to assess the effect of the treatment on each sperm parameter at each incubation time. When p<0.05, the post-hoc analysis was performed by pairwise paired t-tests with Holm’s correction. After 24 hr of storage, LC yielded significantly higher percentages of TM and PM than control, while LC/AC showed better PM. At 48 hr, treated groups showed significantly better motility parameters than control, except AC, where VSL was similar to control and LC/AC. Although no differences were found in flow cytometric parameters, it was noted that treated groups were numerically greater then control in some parameters, and probably with a larger number of ejaculates this numerical trend can be
L-carnitine (LC) and acetyl-L-carnitine (AC) modulate several sperm metabolic functions, such as fatty acid oxidation, acetyl-CoA/free CoA ratio and utilization of pyruvate and lactate as energy substrates. LC has a powerful antioxidant effect by reducing the availability of lipids to peroxidation and increasing activity of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase. Moreover, the presence of carnitines ensures the operation of oxidative pathways by reducing acetylCoA levels and provides acetyl groups for sperm motility. The aim of this study was to evaluate the effect of these substances on cooled sperm viability. Two ejaculates from 6 stallions were diluted with skim milk-based extender (Botu-SemenTM) to a final concentration of 50x106 spermatozoa/mL and then divided into 4 groups: Control (no LC/AC), LC (0.1mM/mL of LC), AC (0.1mM/mL of AC) and LC/ AC (0.1mM/mL of LC+0.1mM/mL of AC). All groups had the osmolarity adjusted to 380 mOsm and pH at 6.8. After treatment, samples were placed at 5 C for 24 and 48 hr. Sperm motility was determined by computer-assisted semen analysis (Hamilton-ThorneTM) for total motility Table 1. Mean values of TM, PM, VSL and RAP analyzed at 0, 24 and 48 hr.
TM PM VSL RAP a,b
0H
24H
C
C
LC
AC
LC/AC
C
48H LC
AC
LC/AC
68.912.7 33,810.2 94,710.7 62.114.0
50.316.2a 2512.6a 86.89.5 41.414.3
59.112.1b 3314.9b 92.614.5 49.014.4
53.417.6a 29.814.0a 89.810.6 45.516.7
56.814.6a 33.312.8b 91.811.5 47.512.1
32.216.5a 12.19.7a 72.113.7a 23.715.6a
45.017.5b 22.814.3b 84.616.3b 35.417.5b
40.316.7b 1913.2c 77.615.3a,c 30.116.9b
43.717.6b 21.613.5b,c 82.215.4b,c 33.416.8b
Values in the same row with different superscript differ significantly (p<0.05).
(TM), progressive motility (PM), average path velocity (VAP), straight line velocity (VSL), curvilinear path velocity (VCL) and rapid sperm (RAP). Plasma membrane and
* Presenting author E-mail address: [email protected] (F.L. Lisboa).
confirmed. Based on these results, it was concluded that the addition of L-carnitine and Acetyl-L-Carnitine improves the maintenance of sperm motility after 24 hr storage at 5 C.