- Email: [email protected]
Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid
Journal of Equine Veterinary Science 32 (2012) 289-291
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Short Communication
Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid Katheryn L. Cerny BS a , Sydney Hughes BS a, Juliana R. Campos DVM b, Robert J. Coleman PhD c, Mats H.T. Troedsson DVM, PhD, ACT a, Edward L. Squires MS, PhD, ACT (Hon) a a b c
Department of Veterinary Science, Equine Reproduction, Maxwell H Gluck Equine Research Center, University of Kentucky, Lexington, KY Department of Veterinary Science, Virology, Maxwell H Gluck Equine Research Center, University of Kentucky, Lexington, KY Department of Animal and Food Sciences, University of Kentucky, 900 W.P Garrigus Building, Lexington, KY
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 July 2011 Received in revised form 25 August 2011 Accepted 27 September 2011 Available online 22 December 2011
The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- 0.5-mL straws (200 106/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 106 21, 59 106 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozenthawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Stallion Spermatozoa Single Layer Centrifugation Cryopreservation
1. Introduction Freezing and thawing causes major damage to the spermatozoa because of factors such as unequal distribution of cryoprotectants, osmotic stress during dehydration and rehydration, phase transitions in the plasma membrane, and oxidative damage [1]. Freezeethaw damage to the spermatozoa results in lower motility, and sperm may be more susceptible to premature capacitation [2]. Also, variation between individual stallions in cryosurvival poses Corresponding author at: K.L. Cerny, BS, Department of Veterinary Science, Maxwell H. Gluck Equine Research, University of Kentucky, Lexington, KY 40546-0099. E-mail address: [email protected] (K.L. Cerny). 0737-0806/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2011.09.075
a challenge to development of freezing protocols that result in high fertility [3]. The increasing use of artificial insemination with frozen-thawed stallion semen necessitates efforts to improve its quality and fertility. Single layer centrifugation (SLC) uses a density gradient to separate spermatozoa with differing specific gravities, resulting in a subpopulation of spermatozoa with increased quality. Numerous studies have demonstrated centrifugation of fresh, cooled, and frozen-thawed semen through a single layer of colloid improves sperm parameters [4-7]. Recent studies using SLC with frozen-thawed stallion semen showed improved sperm motility, chromatin structure, membrane integrity, and mitochondrial membrane potential [6e8]. However, studies on fertility of mares inseminated with sperm centrifuged through a single layer of
290
K.L. Cerny et al. / Journal of Equine Veterinary Science 32 (2012) 289-291
colloid have only been reported for fresh semen or cooled semen from problem stallions [9,10]. One study found pregnancy rates per cycle were increased after insemination of spermatozoa that were selected by gradient centrifugation (44 of 52; 62%) versus simple centrifugation (30 of 52; 42%) [9]. The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by SLC through a colloid before insemination, using a stallion with proven fertility. In addition, changes in semen parameters, including motility, were determined before and after SLC. The hypothesis was that frozen-thawed spermatozoa centrifuged through a single layer of colloid would result in a higher pregnancy rate compared with frozen-thawed spermatozoa that was not centrifuged. 2. Materials and Methods 2.1. Animals Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- 0.5-mL straws (200 106/mL) of frozen-thawed semen. Treatment mares were inseminated with frozen-thawed sperm after processing by SLC. Mares were examined by transrectal ultrasonography to determine stage of the estrous cycle. Mares with a corpus luteum on the ovary were injected with prostaglandin F2alpha (PGF2a) to induce estrus, and those with no corpus luteum were examined every 3 days until estrus was detected by ultrasonographic examination of the uterus for edema. Once mares were confirmed to have at least one spontaneous ovulation and were in mid-diestrus, they were given PGF2a to induce another estrus. Estrous mares with a 35-mm follicle and uterine edema were given 1.5-mg deslorelin intramuscularly (BET Pharmacy, Lexington, KY) to induce ovulation and standardize time of insemination. At the time of deslorelin treatment, mares were assigned to one of two insemination groups: control or SLC-sperm. Thirty-six hours after deslorelin administration, the presence of a preovulatory follicle was confirmed, and mares were inseminated deep into the uterine horn ipsilateral to the ovary containing the ovulatory follicle by using Minitube IUI pipettes (Minitube of America, Verona, WI). Ovulation was confirmed 12 hours after insemination. If a mare did not ovulate within 12 hours after insemination, the cycle was not included in the results, and the mare was given PGF2a to induce another estrus. Mares were examined with ultrasonography for pregnancy on days 12 and 14 after ovulation. All mares were given PGF2a at day 14, and on return to estrus, they were assigned to the alternate treatment until each mare was bred on three cycles. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test.
collected using a Missouri model artificial vagina equipped with an inline filter to separate the gel fraction. Semen was extended 1:3 with EquiPro equine semen extender (Minitube of America, Verona, WI) at 37 C. To assess total progressive motility before further processing, a sample was taken from the extended semen and diluted with EquiPro equine semen extender (Minitube) at 37 C to reach a concentration of 20 106/mL. A 10-mL aliquot was pipetted onto a 37 C glass slide (Minitube of America, Verona, WI), and an 18 18-mm2 cover slip (Minitube of America, Verona, WI) was placed on top. Computer assisted sperm analysis was performed using the SpermVision (Minitube of America, Verona, WI) software program on a 37 C stage. Extended semen was then centrifuged in a 50-mL conical centrifuge tube for 10 minutes at 600 g. After centrifugation, the supernatant was removed, leaving approximately 10% seminal plasma. Volume of seminal plasma left in semen was calculated by estimating the total volume of semen and extender to freeze and multiplying by 10%. Volume of EquiPro Cryoguard complete freezing extender (Minitube of America, Verona, WI) was estimated using the initial concentration of the semen and was added accordingly to reach a concentration of 200 106/mL. The final concentration of 200 106/mL was determined using a hemacytometer (Animal Reproduction Systems, Chino, CA). Extended semen was then loaded into 0.5-mL straws and frozen using IceCube automatic freezer (Minitube of America, Verona, WI). Freezing rate was 10 C/min from 20 C to 15 C and then 25 C/min from 15 C to 120 C, at which point, straws were plunged into liquid nitrogen [11]. After straws were frozen, they were stored in a liquid nitrogen container until further use. To further confirm concentration, spermatozoa were counted using a Nucleocounter SP-100 (Chemometec, Allerod, Denmark) after freezing and thawing. 2.3. SLC and Semen Analysis Four 0.5-mL straws (400 106 spermatozoa) were thawed in a water bath at 37 C for 30 seconds, and the contents were transferred into a tube that was also warmed to 37 C. A 10-mL sample was taken and extended to 20 106/mL to evaluate motility. One milliliter of EquiPro Cryoguard freezing extender (Minitube) was added to the thawed semen to reach a final volume of 3 mL. Four milliliters of EquiPure Bottom Layer (Nidacon, Molndal, Sweden) was transferred to two 15-mL conical centrifuge tubes, and 1.5 mL of extended thawed semen was layered on top. Tubes were then centrifuged for 30 minutes at 300 g at room temperature. After centrifugation, the sperm pellets were combined and resuspended in EquiPro Cryoguard freezing extender (Minitube) to a reach a final volume of 2 mL. A 20-mL sample was taken to evaluate postSLC spermatozoa motility and concentration using SpermVision (Minitube) and Nucleocounter SP-100 (Chemometec). Mares were then inseminated with the SLC sperm.
2.2. Semen Processing and Freezing
3. Results
For semen cryopreservation, five collections were processed during the 2010 breeding season. Semen was
Centrifugation of sperm through a single layer of colloid resulted in a 21.8% recovery of spermatozoa; thus, the
K.L. Cerny et al. / Journal of Equine Veterinary Science 32 (2012) 289-291
291
Table 1 Pregnancy rates at day 14, motility of frozen-thawed semen and SLC sperm, and sperm recovery after SLC Groups
Starting dose volume/total number
Pregnancy rates %
% Progressively motile
Sperm % recovery
% Progressively motile sperm inseminated
Control SLC sperm
2 mL: 411.2 106 24.6 2 mL: 411.2 106 24.6
60% (18/30) 60% (18/30)
41.6 4.2a 67.7 9.2b
N/A 21.8 10.4
171 106 21 59 106 25
SLC, single layer centrifugation. Pregnancy at day 14 did not differ statistically significant (P ¼ 1). a,b Means in a column with different superscripts differ P < .05.
control mares were inseminated with 171 106 21 progressively motile sperm, and treatment mares were inseminated with 59 106 25 progressively motile sperm. The number of progressively motile sperm inseminated was greater in control mares than that in the mares inseminated with SLC sperm (P < .05). The percent progressively motile sperm was improved with SLC (P <.05). The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen. (Table 1) 4. Discussion and Conclusions Four 0.5-mL straws (400 106 spermatozoa) were used in this study instead of the usual 8 straw insemination dose for frozen-thawed semen. This was done in an attempt to more easily detect a difference in pregnancy rates using a relatively small number of mares. Previous studies have found improved pregnancy rates with problem stallions after SLC treatment, but only fresh or cooled semen was used [9,10] The original hypothesis that SLC processing would improve pregnancy rates was not supported. Apparently, a dose of 59 106 25 progressively motile sperm with this stallion was sufficient to achieve normal fertility with frozen-thawed spermatozoa. This may indicate that SLC treatment of semen from a fertile stallion with good semen quality may not be justified. Alternatively, the sperm numbers in the two groups combined with the small number of mares may have prevented us from showing a true difference in fertility. Further studies are needed with more stallions having variable semen quality. Another view would be that SLC processing of frozen-thawed semen did not decrease pregnancy rates even though sperm numbers in the SLC group were approximately one-third of the controls. Because number of motile sperm in this study was not standardized between groups, we were not able to test whether the increased presence of nonmotile, damaged sperm in the control group had any adverse affect. However, the control doses did contain a higher number of nonmotile, damaged, or functionally abnormal sperm compared with the SLC-processed doses. It can be debated that increased presence of the nonmotile, damaged sperm in the control doses did not adversely affect fertility rates. The recovery of sperm after SLC of 22% was similar to what has been published previously [3,4]. Studies are needed to improve sperm recovery after SLC. This might include changing the volume of gradient, tube size, and/or centrifugation speed and time. If recoveries could be increased, the numbers of mares bred to a SLC-processed dose could be increased. Progressive motility after SLC
processing was 26% higher than that from the control semen. Other researchers have reported increases in sperm parameters after SLC of fresh, cooled, and frozen-thawed semen as well [4-7]. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination. Acknowledgments The authors thank Cecilia Hylton and the Cecil and Irene Hylton Foundation for funding this study. They also thank Dr. Ball, Dr. Klein, Dr. Scoggins, Dr. Petersen, Dr. Campos, Lauren Keith, Kevin Gallagher, and Chad Tucker for their help and support, and Hagyards Equine Medical Institute for the use of their nucleocounter. References [1] Squires EL, Keith SL, Graham JK. Evaluation of alternative cryoprotectants for preserving stallion spermatozoa. Theriogenology 2004;62:1056-65. [2] O’Connell M, McClure N, Lewis S. The effects of cryopreservation on sperm morphology, motility and mitochondrial function. Human Reprod 2002;17:704-9. [3] Loomis PR, Graham JK. Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Anim Reprod Sci 2008;105:119-28. [4] Macpherson M, Blanchard T, Love C, Brinsko S, Thompson J, Varner D. Use of a silane-coated silica particle solution to enhance semen quality of stallions. AAEP Proceedings 2003;49:347-9. [5] Morrell J, Dalin A, Rodriguez-Martinez H. Comparison of density gradient and single layer centrifugation of stallion spermatozoa: yield, motility and survival. Equine Vet J 2009;41:53-8. [6] Morrell J, Garcia M, Pena F, Johannisson A. Processing stored stallion semen doses by single layer centrifugation. Theriogenology 2011;76(8):1424-32. [7] Garcia M, Morrell J, Ortega-Ferrusola C, Gonzalez-Fernandez L, Tapia J, Rodriguez-Martinez H, et al. Centrifugation on a single layer of colloid selects improved quality spermatozoa from frozenthawed stallion semen. Anim Reprod Sci 2009;114:193-202. [8] Garcia M, Gonzalez-Fernandez L, Morrell J, Ortega-Ferrusola C, Tapia J, Rodriguez-Martinez H, et al. Single-layer centrifugation through colloid positively modifies the sperm subpopulation structure of frozen-thawed stallion spermatozoa. Reprod Domest Anim 2009;44:523-6. [9] Mari G, Castagnetti C, Morganti M, Rizzato G, Mislei B, Iacono E, et al. Comparison of density gradient and simple centrifugation of equine spermatozoa: effect on fertility of an oligospermic-subfertile stallion. Anim Reprod Sci 2010;121:153-4. [10] Morrell J, Mari G, Kutvolgyi G, Meurling S, Mislei B, Iacono E, et al. Pregnancies following artificial insemination with spermatozoa from problem stallion ejaculates processed by single layer centrifugation with Androcoll-E. Reprod Domest Anim 2011;46:642-5. [11] Cristanelli MJ, Squires EL, Amann RP, Pickett BW. Fertility of stallion semen processed, frozen and thawed by a new procedure. Theriogenology 1984;22:39-45.
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Short Communication
Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid Katheryn L. Cerny BS a , Sydney Hughes BS a, Juliana R. Campos DVM b, Robert J. Coleman PhD c, Mats H.T. Troedsson DVM, PhD, ACT a, Edward L. Squires MS, PhD, ACT (Hon) a a b c
Department of Veterinary Science, Equine Reproduction, Maxwell H Gluck Equine Research Center, University of Kentucky, Lexington, KY Department of Veterinary Science, Virology, Maxwell H Gluck Equine Research Center, University of Kentucky, Lexington, KY Department of Animal and Food Sciences, University of Kentucky, 900 W.P Garrigus Building, Lexington, KY
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 July 2011 Received in revised form 25 August 2011 Accepted 27 September 2011 Available online 22 December 2011
The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- 0.5-mL straws (200 106/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 106 21, 59 106 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozenthawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Stallion Spermatozoa Single Layer Centrifugation Cryopreservation
1. Introduction Freezing and thawing causes major damage to the spermatozoa because of factors such as unequal distribution of cryoprotectants, osmotic stress during dehydration and rehydration, phase transitions in the plasma membrane, and oxidative damage [1]. Freezeethaw damage to the spermatozoa results in lower motility, and sperm may be more susceptible to premature capacitation [2]. Also, variation between individual stallions in cryosurvival poses Corresponding author at: K.L. Cerny, BS, Department of Veterinary Science, Maxwell H. Gluck Equine Research, University of Kentucky, Lexington, KY 40546-0099. E-mail address: [email protected] (K.L. Cerny). 0737-0806/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2011.09.075
a challenge to development of freezing protocols that result in high fertility [3]. The increasing use of artificial insemination with frozen-thawed stallion semen necessitates efforts to improve its quality and fertility. Single layer centrifugation (SLC) uses a density gradient to separate spermatozoa with differing specific gravities, resulting in a subpopulation of spermatozoa with increased quality. Numerous studies have demonstrated centrifugation of fresh, cooled, and frozen-thawed semen through a single layer of colloid improves sperm parameters [4-7]. Recent studies using SLC with frozen-thawed stallion semen showed improved sperm motility, chromatin structure, membrane integrity, and mitochondrial membrane potential [6e8]. However, studies on fertility of mares inseminated with sperm centrifuged through a single layer of
290
K.L. Cerny et al. / Journal of Equine Veterinary Science 32 (2012) 289-291
colloid have only been reported for fresh semen or cooled semen from problem stallions [9,10]. One study found pregnancy rates per cycle were increased after insemination of spermatozoa that were selected by gradient centrifugation (44 of 52; 62%) versus simple centrifugation (30 of 52; 42%) [9]. The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by SLC through a colloid before insemination, using a stallion with proven fertility. In addition, changes in semen parameters, including motility, were determined before and after SLC. The hypothesis was that frozen-thawed spermatozoa centrifuged through a single layer of colloid would result in a higher pregnancy rate compared with frozen-thawed spermatozoa that was not centrifuged. 2. Materials and Methods 2.1. Animals Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- 0.5-mL straws (200 106/mL) of frozen-thawed semen. Treatment mares were inseminated with frozen-thawed sperm after processing by SLC. Mares were examined by transrectal ultrasonography to determine stage of the estrous cycle. Mares with a corpus luteum on the ovary were injected with prostaglandin F2alpha (PGF2a) to induce estrus, and those with no corpus luteum were examined every 3 days until estrus was detected by ultrasonographic examination of the uterus for edema. Once mares were confirmed to have at least one spontaneous ovulation and were in mid-diestrus, they were given PGF2a to induce another estrus. Estrous mares with a 35-mm follicle and uterine edema were given 1.5-mg deslorelin intramuscularly (BET Pharmacy, Lexington, KY) to induce ovulation and standardize time of insemination. At the time of deslorelin treatment, mares were assigned to one of two insemination groups: control or SLC-sperm. Thirty-six hours after deslorelin administration, the presence of a preovulatory follicle was confirmed, and mares were inseminated deep into the uterine horn ipsilateral to the ovary containing the ovulatory follicle by using Minitube IUI pipettes (Minitube of America, Verona, WI). Ovulation was confirmed 12 hours after insemination. If a mare did not ovulate within 12 hours after insemination, the cycle was not included in the results, and the mare was given PGF2a to induce another estrus. Mares were examined with ultrasonography for pregnancy on days 12 and 14 after ovulation. All mares were given PGF2a at day 14, and on return to estrus, they were assigned to the alternate treatment until each mare was bred on three cycles. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test.
collected using a Missouri model artificial vagina equipped with an inline filter to separate the gel fraction. Semen was extended 1:3 with EquiPro equine semen extender (Minitube of America, Verona, WI) at 37 C. To assess total progressive motility before further processing, a sample was taken from the extended semen and diluted with EquiPro equine semen extender (Minitube) at 37 C to reach a concentration of 20 106/mL. A 10-mL aliquot was pipetted onto a 37 C glass slide (Minitube of America, Verona, WI), and an 18 18-mm2 cover slip (Minitube of America, Verona, WI) was placed on top. Computer assisted sperm analysis was performed using the SpermVision (Minitube of America, Verona, WI) software program on a 37 C stage. Extended semen was then centrifuged in a 50-mL conical centrifuge tube for 10 minutes at 600 g. After centrifugation, the supernatant was removed, leaving approximately 10% seminal plasma. Volume of seminal plasma left in semen was calculated by estimating the total volume of semen and extender to freeze and multiplying by 10%. Volume of EquiPro Cryoguard complete freezing extender (Minitube of America, Verona, WI) was estimated using the initial concentration of the semen and was added accordingly to reach a concentration of 200 106/mL. The final concentration of 200 106/mL was determined using a hemacytometer (Animal Reproduction Systems, Chino, CA). Extended semen was then loaded into 0.5-mL straws and frozen using IceCube automatic freezer (Minitube of America, Verona, WI). Freezing rate was 10 C/min from 20 C to 15 C and then 25 C/min from 15 C to 120 C, at which point, straws were plunged into liquid nitrogen [11]. After straws were frozen, they were stored in a liquid nitrogen container until further use. To further confirm concentration, spermatozoa were counted using a Nucleocounter SP-100 (Chemometec, Allerod, Denmark) after freezing and thawing. 2.3. SLC and Semen Analysis Four 0.5-mL straws (400 106 spermatozoa) were thawed in a water bath at 37 C for 30 seconds, and the contents were transferred into a tube that was also warmed to 37 C. A 10-mL sample was taken and extended to 20 106/mL to evaluate motility. One milliliter of EquiPro Cryoguard freezing extender (Minitube) was added to the thawed semen to reach a final volume of 3 mL. Four milliliters of EquiPure Bottom Layer (Nidacon, Molndal, Sweden) was transferred to two 15-mL conical centrifuge tubes, and 1.5 mL of extended thawed semen was layered on top. Tubes were then centrifuged for 30 minutes at 300 g at room temperature. After centrifugation, the sperm pellets were combined and resuspended in EquiPro Cryoguard freezing extender (Minitube) to a reach a final volume of 2 mL. A 20-mL sample was taken to evaluate postSLC spermatozoa motility and concentration using SpermVision (Minitube) and Nucleocounter SP-100 (Chemometec). Mares were then inseminated with the SLC sperm.
2.2. Semen Processing and Freezing
3. Results
For semen cryopreservation, five collections were processed during the 2010 breeding season. Semen was
Centrifugation of sperm through a single layer of colloid resulted in a 21.8% recovery of spermatozoa; thus, the
K.L. Cerny et al. / Journal of Equine Veterinary Science 32 (2012) 289-291
291
Table 1 Pregnancy rates at day 14, motility of frozen-thawed semen and SLC sperm, and sperm recovery after SLC Groups
Starting dose volume/total number
Pregnancy rates %
% Progressively motile
Sperm % recovery
% Progressively motile sperm inseminated
Control SLC sperm
2 mL: 411.2 106 24.6 2 mL: 411.2 106 24.6
60% (18/30) 60% (18/30)
41.6 4.2a 67.7 9.2b
N/A 21.8 10.4
171 106 21 59 106 25
SLC, single layer centrifugation. Pregnancy at day 14 did not differ statistically significant (P ¼ 1). a,b Means in a column with different superscripts differ P < .05.
control mares were inseminated with 171 106 21 progressively motile sperm, and treatment mares were inseminated with 59 106 25 progressively motile sperm. The number of progressively motile sperm inseminated was greater in control mares than that in the mares inseminated with SLC sperm (P < .05). The percent progressively motile sperm was improved with SLC (P <.05). The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen. (Table 1) 4. Discussion and Conclusions Four 0.5-mL straws (400 106 spermatozoa) were used in this study instead of the usual 8 straw insemination dose for frozen-thawed semen. This was done in an attempt to more easily detect a difference in pregnancy rates using a relatively small number of mares. Previous studies have found improved pregnancy rates with problem stallions after SLC treatment, but only fresh or cooled semen was used [9,10] The original hypothesis that SLC processing would improve pregnancy rates was not supported. Apparently, a dose of 59 106 25 progressively motile sperm with this stallion was sufficient to achieve normal fertility with frozen-thawed spermatozoa. This may indicate that SLC treatment of semen from a fertile stallion with good semen quality may not be justified. Alternatively, the sperm numbers in the two groups combined with the small number of mares may have prevented us from showing a true difference in fertility. Further studies are needed with more stallions having variable semen quality. Another view would be that SLC processing of frozen-thawed semen did not decrease pregnancy rates even though sperm numbers in the SLC group were approximately one-third of the controls. Because number of motile sperm in this study was not standardized between groups, we were not able to test whether the increased presence of nonmotile, damaged sperm in the control group had any adverse affect. However, the control doses did contain a higher number of nonmotile, damaged, or functionally abnormal sperm compared with the SLC-processed doses. It can be debated that increased presence of the nonmotile, damaged sperm in the control doses did not adversely affect fertility rates. The recovery of sperm after SLC of 22% was similar to what has been published previously [3,4]. Studies are needed to improve sperm recovery after SLC. This might include changing the volume of gradient, tube size, and/or centrifugation speed and time. If recoveries could be increased, the numbers of mares bred to a SLC-processed dose could be increased. Progressive motility after SLC
processing was 26% higher than that from the control semen. Other researchers have reported increases in sperm parameters after SLC of fresh, cooled, and frozen-thawed semen as well [4-7]. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination. Acknowledgments The authors thank Cecilia Hylton and the Cecil and Irene Hylton Foundation for funding this study. They also thank Dr. Ball, Dr. Klein, Dr. Scoggins, Dr. Petersen, Dr. Campos, Lauren Keith, Kevin Gallagher, and Chad Tucker for their help and support, and Hagyards Equine Medical Institute for the use of their nucleocounter. References [1] Squires EL, Keith SL, Graham JK. Evaluation of alternative cryoprotectants for preserving stallion spermatozoa. Theriogenology 2004;62:1056-65. [2] O’Connell M, McClure N, Lewis S. The effects of cryopreservation on sperm morphology, motility and mitochondrial function. Human Reprod 2002;17:704-9. [3] Loomis PR, Graham JK. Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Anim Reprod Sci 2008;105:119-28. [4] Macpherson M, Blanchard T, Love C, Brinsko S, Thompson J, Varner D. Use of a silane-coated silica particle solution to enhance semen quality of stallions. AAEP Proceedings 2003;49:347-9. [5] Morrell J, Dalin A, Rodriguez-Martinez H. Comparison of density gradient and single layer centrifugation of stallion spermatozoa: yield, motility and survival. Equine Vet J 2009;41:53-8. [6] Morrell J, Garcia M, Pena F, Johannisson A. Processing stored stallion semen doses by single layer centrifugation. Theriogenology 2011;76(8):1424-32. [7] Garcia M, Morrell J, Ortega-Ferrusola C, Gonzalez-Fernandez L, Tapia J, Rodriguez-Martinez H, et al. Centrifugation on a single layer of colloid selects improved quality spermatozoa from frozenthawed stallion semen. Anim Reprod Sci 2009;114:193-202. [8] Garcia M, Gonzalez-Fernandez L, Morrell J, Ortega-Ferrusola C, Tapia J, Rodriguez-Martinez H, et al. Single-layer centrifugation through colloid positively modifies the sperm subpopulation structure of frozen-thawed stallion spermatozoa. Reprod Domest Anim 2009;44:523-6. [9] Mari G, Castagnetti C, Morganti M, Rizzato G, Mislei B, Iacono E, et al. Comparison of density gradient and simple centrifugation of equine spermatozoa: effect on fertility of an oligospermic-subfertile stallion. Anim Reprod Sci 2010;121:153-4. [10] Morrell J, Mari G, Kutvolgyi G, Meurling S, Mislei B, Iacono E, et al. Pregnancies following artificial insemination with spermatozoa from problem stallion ejaculates processed by single layer centrifugation with Androcoll-E. Reprod Domest Anim 2011;46:642-5. [11] Cristanelli MJ, Squires EL, Amann RP, Pickett BW. Fertility of stallion semen processed, frozen and thawed by a new procedure. Theriogenology 1984;22:39-45.