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Effect of different sperm concentration methods on sperm quality and recovery rate
482
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518
gonadal status in stallions. Serum samples were collected from newborn (n ¼ 8), yearling (n ¼ 8; 13-15 months), and adult stallions ( 2 years; n¼15), and seasonal changes in AMH were determined based upon monthly samples from 9 stallions. Additional serum samples from geldings (n ¼ 41), cryptorchids (n ¼ 41), and intact stallions (n ¼ 15) were evaluated for serum AMH concentrations. The biological half-life of serum AMH was determined by daily sampling post-castration in three stallions. All serum samples were analyzed for AMH by a commercially available ELISA (Active AMH-ELISA; Diagnostic Systems Laboratories, Webster, TX) that was validated for the stallion. Data were analyzed by ANOVA and are presented as mean S.E.M. Serum AMH concentrations were similar between newborn (26.4 2.1 ng/mL) and yearling colts (25.0 3.5 ng/mL) and decreased (P < 0.02) after puberty (14.7 2.4 ng/mL). Serum concentrations of AMH peaked in May and reached a nadir in November (northern hemisphere). Concentrations of AMH were significantly higher in cryptorchid stallions (32.7 5.3 ng/mL) than in intact stallions (14.7 2.4 ng/ mL) or geldings (0.07 0.01 ng/mL). Finally, serum AMH concentrations declined exponentially post castration, and the calculated biological half-life was 1.5 days. The results of this study indicate that serum AMH concentrations are high at birth and decline significantly after puberty and that there is a significant seasonal variation in serum AMH concentrations with a peak in spring and a nadir in fall. Furthermore, serum AMH concentrations were higher in cryptorchids than in intact stallions or castrates, and determination of serum AMH concentrations may be useful for diagnosis of cryptorchidism in stallions. Acknowledgments Supported by the John P. Hughes Endowment and the Albert G. Clay Endowment
season. The stallions were initially kept for an abstinence period of 10 days after which semen was collected. After a further period of 3 days of abstinence, semen was collected again and fractionated. Semen was collected using an open ended artificial vagina (Missouri model with open end). The first fraction of the ejaculate (one jet) and second sperm fraction (rest of the ejaculate) were obtained in separated vials and extended in INRA96. They were immediately evaluated for sperm concentration, sperm motility using a computerized analysis system (SCA), sperm morphology, and sperm DNA fragmentation using the Halomax kit at time 0 (SDF-t0) and after 6 (r-SDF-t6) and 24 (r-SDF-24h) hours of incubation in a water bath at 37oC. Significant differences in sperm concentration, sperm morphology, SDF-t0 and r-SDF-t6 between fractions were found. However, no significant differences in sperm motility and r-SDF-t24 were seen. The first fraction exhibited higher motility, less morphological anomalies and lower SDF-t0 and SDF-t6 than the second fraction. When significant differences between fractions in both ejaculates of each stallion were compared, sperm concentration was on average 57% (ejaculate 1) and 64% (ejaculate 2) higher in the first fraction of the ejaculate than in the second. Regarding sperm morphology, morphologically normal sperm were 39% higher in the first fraction than in the second. r-SDF-T0 was lower in the first fraction in both ejaculates of each stallion, but this reduction was lower in the second ejaculate comparing to the first (-53% versus -102%). r-SDF-t6 was also lower in the first fraction of the ejaculate, but in this case the reduction was higher in the second ejaculate (-17% versus -180%). Sperm characteristics of the first ejaculated fraction in stallion, especially those concerning the level of DNA damage, showed the most efficient values to achieve an efficient pregnancy. When we mix both fractions in a regular ejaculation for sperm extension and ex vivo handling of the sperm samples, we are diluting the whole potential benefit accumulated in the first fraction of the ejaculate.
Impact on sperm characteristics of ejaculate fractionation and collection interval in stallions F. Crespo 1, J.C. Blázquez 1, C. López-Fernández 2, and J. Gosálvez 2 1 Centro Militar de Cría Caballar: FESCCR-Ministry of Defense, Ávila, Spain, 2 Biology Department, Genetic Unit, UAM, Madrid, Spain Different strategies are available to improve the semen characteristics obtained in the original ejaculate. Most of these techniques, such as swim up or density gradient centrifugation, are conducted for ex vivo selection of spermatozoa. It has also been demonstrated recently in human donors and patients that sperm DNA fragmentation improves with recurrent ejaculation, and conversely prolonged sexual abstinence can reduce semen quality. The aim of this study was to analyze stallion sperm quality observed in two sequential fractions produced in an ejaculation in order to explore whether sperm recovered later during ejaculation had improved sperm characteristics compared to the initial fraction. Six stallions (two ejaculates per stallion) were analyzed during the breeding
Effect of different sperm concentration methods on sperm quality and recovery rate G.M. Davolli, G.C. Camozzato, M.M. Figueiredo, H.B.A. Bastos, N. Cazales, and R.C. Mattos REPROLAB, Faculdade de Veterinária, UFRGS, Porto Alegre, Brasil The ability to increase the concentration of sperm is important to process sperm for cooling and freezing. The available techniques are also useful, especially with semen of poor quality, for separating spermatozoa from seminal plasma and extender, concentrating sperm for freezing and cooling, and for low-dose insemination. At the present moment, this concentration is usually done by centrifugation. However, centrifugation subjects the sperm to a certain amount of stress, more in some stallions than others. Filtration of semen was also proposed as a concentrating method, and it was thought that it would result in less sperm injury. The objective of this study was to compare centrifugation, cushioned centrifugation and
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518
semen filtration for their sperm retention efficiency and their effects on sperm quality parameters. Fifteen ejaculates from four stallions were collected and immediately extended with skim milk 1:1 (v/v). Extended semen was adjusted to a final concentration up to 50 x 106 sptz/mL using a Neubauer chamber. Three aliquots of each ejaculate were submitted to the following procedures: (a - Cent) centrifugation of 20 mL of extended semen in a 50 mL conical tube at 600 x g for 10 min, (b - Cushion) centrifugation at 1000 x g for 20 min of 20 mL of extended semen in a 50 mL conical tube with 1 mL of cushion solution (EqcellsireÒ component B, IMV, France) at the bottom, and (c - Filter) filtration with commercial filter (Sperm FilterÒ, Botupharma, Brazil) with a hydrophilic synthetic membrane that by gravity allows the seminal plasma to pass through and not the sperm, due to pore diameter. Following centrifugation supernate was aspirated and, when present, the cushion fluid was removed. Sperm pellets and filtered semen was resuspended with skim milk at 25 x 106 sperm/mL and immediately evaluated for total motility, progressive motility, velocity, membrane funcionality (HOST) and membrane integrity (CFDA/PI). Data were analyzed by one-way ANOVA and means compared by the Tukey test at 5% level of significance. The sperm recovery rate of each method was calculated dividing the total number of sperm recovered by the initial total sperm number of each aliquot. The results are shown in Table 1.
Table 1 Means ( SD). Effect of different concentrating methods on sperm quality and sperm recovery rate Cent Total motility (%) 56 Progressive motility (%) 27 Velocity (1-5) 2.3 Membrane Functionality (%) 62 Membrane Integrity (%) 67 Sperm Recovery (%) 72a a,b
Cushion 26 14 0.9 7 14 11
59 28 2.2 64 72 85b
P (Value)
Filter
27 54 14 38 0.9 2.4 8 68 17 65 18 77ab
26 23 0.6 8 14 14
0.59 0.18 0.54 0.15 0.52 0.05
Different superscripts in each row differ significantly (P 0.05).
The results in Table 1 show no significant difference comparing the different methods for total motility, progressive motility, sperm velocity, membrane functionality and membrane integrity. Regarding the sperm recovery rates, there was a significant difference between conventional centrifugation and centrifugation using cushion solution at the bottom of the tube. Based on our results, we conclude that centrifugation with cushion is
483
more advantageous than conventional centrifugation, although it did not show a better recovery rate compared to filtration. Seasonal effects on behavioral traits of stabled Criollo stallions during semen collection D.C. de Souza 1, S.D. Schiavo 1, M.A. Peres Maciel 1, N.M. Fajardo Campos 1, S.I. dos Santos 1, I.C. Bustamante-Filho 3, L.S. Van der Linden 2, S.F. Rechsteiner 2, and A. Pires Neves 1, 2 1 UNIPAMPA (Universidade Federal do Pampa (Dom Pedrito, RS, Brazil), 2 Programa de Pós-Graduação em Medicina Animal: Equinos (UFRGS) – Porto Alegre, RS, Brazil, 3 Centro Universitário UNIVATES, Lajeado, Brazil
The Criollo horse is in the third position among the horse breed registrations in Brazil. However, most of the behavioral traits in this breed are unknown because the breeders keep their animals free in the pasture. Recently, the Association of Criollo Breeders authorized the use of artificial insemination and embryo transfer. This study investigates sexual behavior during semen collections. Fifteen Criollo stallions kept in individual stables in horse farms in Dom Pedrito and Lavras do Sul, Rio Grande do Sul, Brazil were used. Each stallion was collected one to six times using on an estrous mare, and the collections were recorded by a Canon SX210 camera. The video images were analyzed according to the following parameters: time to react (TR) was the time from approaching the mare to achievement of erection; time to mount (TM) was the period between erection and the first mount, time to ejaculate (TE) was the period between penetration and ejaculation. All the stallions behaved normally during the semen collections sessions during all months, but the TR seemed to be shorter during non-breeding season. During the semen collection sessions, 70% of the stallions vocalized, but 80% did not show the flehmen reflex. These results indicate that Criollo stallions exhibit similar sexual behavior as other horse breeds during hand-mating.
Acknowledgments The authors kindly acknowledge the horse farms Estância Santa Anna, Cabanha Dom Marcelino and, Cabanha A Tala which spared their animals for this study. Also, to Luciana Meirelles Richer, DVM, for the suggestions on revision.
Table 1 Time to reaction (TR); time to mount (TM) and time to ejaculate (TE): means, standard deviation and coefficient of variation (CV) Time (seconds)
June-August (Non-Breeding Season)
September (Breeding Season)
April (Non-Breeding Season)
TR TM TE
9.98 3.24CV ¼ 0.32 23.32 15.83CV ¼ 0.68 15.78 3.2CV ¼ 0.2
19.52 13.32CV ¼ 0.68 29.76 15.8CV ¼ 0.53 17.03 4.87CV ¼ 0.29
11.24 4.55CV ¼ 0.4 26.41 9.44CV ¼ 0.36 17.81 5.08CV ¼ 0.29
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518
gonadal status in stallions. Serum samples were collected from newborn (n ¼ 8), yearling (n ¼ 8; 13-15 months), and adult stallions ( 2 years; n¼15), and seasonal changes in AMH were determined based upon monthly samples from 9 stallions. Additional serum samples from geldings (n ¼ 41), cryptorchids (n ¼ 41), and intact stallions (n ¼ 15) were evaluated for serum AMH concentrations. The biological half-life of serum AMH was determined by daily sampling post-castration in three stallions. All serum samples were analyzed for AMH by a commercially available ELISA (Active AMH-ELISA; Diagnostic Systems Laboratories, Webster, TX) that was validated for the stallion. Data were analyzed by ANOVA and are presented as mean S.E.M. Serum AMH concentrations were similar between newborn (26.4 2.1 ng/mL) and yearling colts (25.0 3.5 ng/mL) and decreased (P < 0.02) after puberty (14.7 2.4 ng/mL). Serum concentrations of AMH peaked in May and reached a nadir in November (northern hemisphere). Concentrations of AMH were significantly higher in cryptorchid stallions (32.7 5.3 ng/mL) than in intact stallions (14.7 2.4 ng/ mL) or geldings (0.07 0.01 ng/mL). Finally, serum AMH concentrations declined exponentially post castration, and the calculated biological half-life was 1.5 days. The results of this study indicate that serum AMH concentrations are high at birth and decline significantly after puberty and that there is a significant seasonal variation in serum AMH concentrations with a peak in spring and a nadir in fall. Furthermore, serum AMH concentrations were higher in cryptorchids than in intact stallions or castrates, and determination of serum AMH concentrations may be useful for diagnosis of cryptorchidism in stallions. Acknowledgments Supported by the John P. Hughes Endowment and the Albert G. Clay Endowment
season. The stallions were initially kept for an abstinence period of 10 days after which semen was collected. After a further period of 3 days of abstinence, semen was collected again and fractionated. Semen was collected using an open ended artificial vagina (Missouri model with open end). The first fraction of the ejaculate (one jet) and second sperm fraction (rest of the ejaculate) were obtained in separated vials and extended in INRA96. They were immediately evaluated for sperm concentration, sperm motility using a computerized analysis system (SCA), sperm morphology, and sperm DNA fragmentation using the Halomax kit at time 0 (SDF-t0) and after 6 (r-SDF-t6) and 24 (r-SDF-24h) hours of incubation in a water bath at 37oC. Significant differences in sperm concentration, sperm morphology, SDF-t0 and r-SDF-t6 between fractions were found. However, no significant differences in sperm motility and r-SDF-t24 were seen. The first fraction exhibited higher motility, less morphological anomalies and lower SDF-t0 and SDF-t6 than the second fraction. When significant differences between fractions in both ejaculates of each stallion were compared, sperm concentration was on average 57% (ejaculate 1) and 64% (ejaculate 2) higher in the first fraction of the ejaculate than in the second. Regarding sperm morphology, morphologically normal sperm were 39% higher in the first fraction than in the second. r-SDF-T0 was lower in the first fraction in both ejaculates of each stallion, but this reduction was lower in the second ejaculate comparing to the first (-53% versus -102%). r-SDF-t6 was also lower in the first fraction of the ejaculate, but in this case the reduction was higher in the second ejaculate (-17% versus -180%). Sperm characteristics of the first ejaculated fraction in stallion, especially those concerning the level of DNA damage, showed the most efficient values to achieve an efficient pregnancy. When we mix both fractions in a regular ejaculation for sperm extension and ex vivo handling of the sperm samples, we are diluting the whole potential benefit accumulated in the first fraction of the ejaculate.
Impact on sperm characteristics of ejaculate fractionation and collection interval in stallions F. Crespo 1, J.C. Blázquez 1, C. López-Fernández 2, and J. Gosálvez 2 1 Centro Militar de Cría Caballar: FESCCR-Ministry of Defense, Ávila, Spain, 2 Biology Department, Genetic Unit, UAM, Madrid, Spain Different strategies are available to improve the semen characteristics obtained in the original ejaculate. Most of these techniques, such as swim up or density gradient centrifugation, are conducted for ex vivo selection of spermatozoa. It has also been demonstrated recently in human donors and patients that sperm DNA fragmentation improves with recurrent ejaculation, and conversely prolonged sexual abstinence can reduce semen quality. The aim of this study was to analyze stallion sperm quality observed in two sequential fractions produced in an ejaculation in order to explore whether sperm recovered later during ejaculation had improved sperm characteristics compared to the initial fraction. Six stallions (two ejaculates per stallion) were analyzed during the breeding
Effect of different sperm concentration methods on sperm quality and recovery rate G.M. Davolli, G.C. Camozzato, M.M. Figueiredo, H.B.A. Bastos, N. Cazales, and R.C. Mattos REPROLAB, Faculdade de Veterinária, UFRGS, Porto Alegre, Brasil The ability to increase the concentration of sperm is important to process sperm for cooling and freezing. The available techniques are also useful, especially with semen of poor quality, for separating spermatozoa from seminal plasma and extender, concentrating sperm for freezing and cooling, and for low-dose insemination. At the present moment, this concentration is usually done by centrifugation. However, centrifugation subjects the sperm to a certain amount of stress, more in some stallions than others. Filtration of semen was also proposed as a concentrating method, and it was thought that it would result in less sperm injury. The objective of this study was to compare centrifugation, cushioned centrifugation and
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518
semen filtration for their sperm retention efficiency and their effects on sperm quality parameters. Fifteen ejaculates from four stallions were collected and immediately extended with skim milk 1:1 (v/v). Extended semen was adjusted to a final concentration up to 50 x 106 sptz/mL using a Neubauer chamber. Three aliquots of each ejaculate were submitted to the following procedures: (a - Cent) centrifugation of 20 mL of extended semen in a 50 mL conical tube at 600 x g for 10 min, (b - Cushion) centrifugation at 1000 x g for 20 min of 20 mL of extended semen in a 50 mL conical tube with 1 mL of cushion solution (EqcellsireÒ component B, IMV, France) at the bottom, and (c - Filter) filtration with commercial filter (Sperm FilterÒ, Botupharma, Brazil) with a hydrophilic synthetic membrane that by gravity allows the seminal plasma to pass through and not the sperm, due to pore diameter. Following centrifugation supernate was aspirated and, when present, the cushion fluid was removed. Sperm pellets and filtered semen was resuspended with skim milk at 25 x 106 sperm/mL and immediately evaluated for total motility, progressive motility, velocity, membrane funcionality (HOST) and membrane integrity (CFDA/PI). Data were analyzed by one-way ANOVA and means compared by the Tukey test at 5% level of significance. The sperm recovery rate of each method was calculated dividing the total number of sperm recovered by the initial total sperm number of each aliquot. The results are shown in Table 1.
Table 1 Means ( SD). Effect of different concentrating methods on sperm quality and sperm recovery rate Cent Total motility (%) 56 Progressive motility (%) 27 Velocity (1-5) 2.3 Membrane Functionality (%) 62 Membrane Integrity (%) 67 Sperm Recovery (%) 72a a,b
Cushion 26 14 0.9 7 14 11
59 28 2.2 64 72 85b
P (Value)
Filter
27 54 14 38 0.9 2.4 8 68 17 65 18 77ab
26 23 0.6 8 14 14
0.59 0.18 0.54 0.15 0.52 0.05
Different superscripts in each row differ significantly (P 0.05).
The results in Table 1 show no significant difference comparing the different methods for total motility, progressive motility, sperm velocity, membrane functionality and membrane integrity. Regarding the sperm recovery rates, there was a significant difference between conventional centrifugation and centrifugation using cushion solution at the bottom of the tube. Based on our results, we conclude that centrifugation with cushion is
483
more advantageous than conventional centrifugation, although it did not show a better recovery rate compared to filtration. Seasonal effects on behavioral traits of stabled Criollo stallions during semen collection D.C. de Souza 1, S.D. Schiavo 1, M.A. Peres Maciel 1, N.M. Fajardo Campos 1, S.I. dos Santos 1, I.C. Bustamante-Filho 3, L.S. Van der Linden 2, S.F. Rechsteiner 2, and A. Pires Neves 1, 2 1 UNIPAMPA (Universidade Federal do Pampa (Dom Pedrito, RS, Brazil), 2 Programa de Pós-Graduação em Medicina Animal: Equinos (UFRGS) – Porto Alegre, RS, Brazil, 3 Centro Universitário UNIVATES, Lajeado, Brazil
The Criollo horse is in the third position among the horse breed registrations in Brazil. However, most of the behavioral traits in this breed are unknown because the breeders keep their animals free in the pasture. Recently, the Association of Criollo Breeders authorized the use of artificial insemination and embryo transfer. This study investigates sexual behavior during semen collections. Fifteen Criollo stallions kept in individual stables in horse farms in Dom Pedrito and Lavras do Sul, Rio Grande do Sul, Brazil were used. Each stallion was collected one to six times using on an estrous mare, and the collections were recorded by a Canon SX210 camera. The video images were analyzed according to the following parameters: time to react (TR) was the time from approaching the mare to achievement of erection; time to mount (TM) was the period between erection and the first mount, time to ejaculate (TE) was the period between penetration and ejaculation. All the stallions behaved normally during the semen collections sessions during all months, but the TR seemed to be shorter during non-breeding season. During the semen collection sessions, 70% of the stallions vocalized, but 80% did not show the flehmen reflex. These results indicate that Criollo stallions exhibit similar sexual behavior as other horse breeds during hand-mating.
Acknowledgments The authors kindly acknowledge the horse farms Estância Santa Anna, Cabanha Dom Marcelino and, Cabanha A Tala which spared their animals for this study. Also, to Luciana Meirelles Richer, DVM, for the suggestions on revision.
Table 1 Time to reaction (TR); time to mount (TM) and time to ejaculate (TE): means, standard deviation and coefficient of variation (CV) Time (seconds)
June-August (Non-Breeding Season)
September (Breeding Season)
April (Non-Breeding Season)
TR TM TE
9.98 3.24CV ¼ 0.32 23.32 15.83CV ¼ 0.68 15.78 3.2CV ¼ 0.2
19.52 13.32CV ¼ 0.68 29.76 15.8CV ¼ 0.53 17.03 4.87CV ¼ 0.29
11.24 4.55CV ¼ 0.4 26.41 9.44CV ¼ 0.36 17.81 5.08CV ¼ 0.29