- Email: [email protected]
Comparative morphological analysis and optimization of CASA parameters in bull, ram, buck and dog
Abstracts / Animal Reproduction Science 169 (2016) 99–135
was performed using electronic statistics software (SPSS 23.0). Results: A significant relation between sperm motility, concentration and the occurrence of NVs was found. More fast progressive: [(6.2% vs. 1.5%), and slow progressive: (32.0% vs. 19.8%) (p < 0.01) spermatozoa were observed in men with class I sperm. Sperm concentration negatively correlated with MSOME quality (24.1 vs. 6.8 Mio/ml, p < 0.01). No correlation between male age and MSOME quality was observed. Although female partners of men without class I sperm were substantially younger (33.3 vs. 34.3 years, p < 0.001), and the number of retrieved and MII oocytes was higher (12.51 vs. 13.53, p < 0.05; 11.01 vs. 11.76, p < 0.05, respectively), FR, BR and TBR were significant lower when injecting only class II or III sperm: FR: 69.3% vs. 74.9%, p < 0.001; BR: 30.6% vs. 34.9%, p < 0.01; TBR: 10.4% vs. 9.04%, p < 0.05. Conclusions: The negative correlation between conventional semen parameters and occurrence of NV strongly indicates that NVs reflect a pathological situation. This finding is substantiated by a negative impact on early embryonic development. http://dx.doi.org/10.1016/j.anireprosci.2016.03.054 P36 Immunohistochemical localization of paraoxonase type 1 in the boar genital tract Barranco 1 ,
˜ 1, Perez-Patino
Isabel Cristina Alejandro Vicente-Carrillo 2 , Manuel Alvarez-Rodriguez 2 , Emilio A. Martinez 1 , Jordi Roca 1,∗ , Heriberto Rodriguez-Martinez 2 1 Department of Medicine & Animal Surgery, University of Murcia, Spain 2 Department of Clinical & Experimental Medicine, Linköping University, Sweden E-mail address: [email protected] (J. Roca).
The boar seminal plasma (SP) is composed of secretions from testis, epididymis, and accessory sex glands. The enzyme paraoxonase type 1 (PON-1), a high-density lipoprotein-associated enzyme present in boar-SP protects sperm membranes against oxidative damage. Its activity differs among boars, ejaculates within boar and also among ejaculate fractions (Barranco et al., Andrology, 3:315–20, 2015). These findings could indicate different sites of PON-1 expression in the boar genital tract. Accordingly, this study characterized the expression of PON-1 in the boar reproductive tract. Tissue samples from testis, epididymis and accessory sex glands of six healthy and fertile boars slaughtered for gene replacement reasons were fixed in buffered formaldehyde, embedded in paraffin blocks, sliced (4 m thickness), mounted on slides and subjected to PON-1-immunohistochemical localization using a rabbit primary polyclonal antibody against this enzyme (ab53193, Abcam, Cambridge, UK). All boars expressed PON-1 in the genital tract. Within testis, immune reaction
117
against PON-1 was detected in the interstitium (specifically in Leydig cells and blood vessels) and in the seminiferous tubules (specifically in spermatogonia and elongated spermatids). PON-1 markedly stained the cytoplasm of the epididymal principal cells as well as the lumen (secretion and spermatozoa). All accessory sexual glands showed PON-1-staining; the prostatic secretory cells showed a predominantly intracytoplasmic staining leaving the nuclear area negative. The seminal vesicles and bulbourethral glands cells showed a membranous staining. There were no differences in PON-1 localization nor subjective intensity among boars. In conclusion, the present study showed a wide distribution of PON-1 throughout boar genital tract, including testes, epididymis and accessory sexual glands. Supported by MINECO, Madrid, and FEDER Funds, EU (AGL2012-399903); and Seneca Foundation (19892/GERM/15), Murcia, Spain; FORSS and The Swedish Research Councils VR and FORMAS, Stockholm, Sweden. http://dx.doi.org/10.1016/j.anireprosci.2016.03.055 P37 Comparative morphological analysis and optimization of CASA parameters in bull, ram, buck and dog K. Tekin 1,∗ , C. Stelletta 2 , F. Oztutar Stelletta 1 , A. Daskin 1 1 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Ankara University, Turkey 2 Department of Animal Medicine, Production and Health, University of Padova, Italy E-mail address: [email protected] (K. Tekin).
The aim of the study was to optimize the morphological parameters of the CASA system SCA® (Microptic, Barcelona, Spain), comparing with semi-automatic system LAS® (Leica Microsystems, Wetzlar, Germany) in different species. A total of 60 cryopreserved semen samples of four different species (bovine, canine, caprine and ovine) were analysed. Five different semen straws, for each species, from 3 different individuals were used. Smears were fixed for 10 min by adding 1 ml SpermBlue fixative except ram (ram fixed with 3% glutaraldehyde) and stained for 15 min with SpermBlue stain. The slides were analysed with SCA and LAS. Twenty randomly chosen spermatozoa, were evaluated with the morphological module of the SCA and LAS. The sperm head area, acrosome, midpiece and were captured randomly in different fields rejecting only those that overlapped. Area of spermatozoon heads (area, length, with and acrosome), midpieces (area, width and angle) with SCA and heads (length and width), acrosomes (length and width), midpiece length were measured with LAS. To clarify whether species had any ‘differences’ according to staining, one-way ANOVA and Tukey test were performed over the data distribution. Spermatozoon head length and width average of bovine, canine, caprine and ovine were 5.46 ± 0.05, 11.0 ± 0.08; 4.51 ± 0.03, 7.2 ± 0.05;
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Abstracts / Animal Reproduction Science 169 (2016) 99–135
4.5 ± 0.2, 9.4 ± 0.46; 5.8 ± 0.03, 10.6 ± 0.05 m with SCA; and 9.1 ± 0.06, 4.6 ± 0.04; 5.9 ± 0.04, 3.7 ± 0.03; 8.2 ± 0.05; 4.037 ± 0.04, 8.4 ± 0.03, 4.7 ± 0.04 m with LAS respectively. Angle of insertion midpiece/head shows significant differences (P < 0.05) between different species. Head, acrosome and midpiece parameters from caprine semen were not significantly different with bovine and canine semen. Evaluating the two different methods of analysis, statistically different data have been found in all species (width and length of the head), particularly for bovine, caprine and ovine semen. In order to implement accuracy and repeatability of the SCA® system, improving the algorithm of analysis for ovine and caprine species, as well as increasing the level of standardization throughout the entire process of analysis, appears to be necessary. http://dx.doi.org/10.1016/j.anireprosci.2016.03.056 P38 Exposure to seminal plasma reduces the duration that epididymal ram spermatozoa exhibit wave motion Jessica P. Rickard 1,∗ , Tamara Leahy 1 , Xavier Druart 2 , Simon P. de Graaf 1 1 School of Life and Environmental Sciences, Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia 2 National Institute for Agricultural Research (INRA), University of Tours, 37380 Nouzilly, France E-mail address: [email protected] (J.P. Rickard).
Wave motion (aka mass motility) is commonly used to assess the initial quality of ram semen immediately following ejaculation. However, relatively little is known regarding the mechanisms which govern this phenomenon. One theory suggests that ejaculate sperm concentration plays a role, yet recent work provides evidence to the contrary (David et al., An Reprod Sci, 161, 2015). We hypothesise that contact with seminal plasma at ejaculation influences the initiation and duration of wave motion. As such, this study aims to document epididymal wave motion and observe the influence of seminal plasma on its duration. Epididymal spermatozoa were obtained from the testes of Merino rams (n = 3) at slaughter via microperfusion. Aliquots (25 l) of spermatozoa were then exposed to 50 l of either a tris-citrate-fructose solution, phosphate buffered saline or seminal plasma (previously collected, thawed and warmed as needed). A fourth aliquot of undiluted spermatozoa was used as a control. Wave motion was assessed (6 l on a warmed slide) and scored as per industry standard (1–5; Evans and Maxwell, Artificial insemination of sheep and goats, 1987) immediately post supplementation and every 2 min thereafter until 10 min (samples maintained at 37 ◦ C). Samples were then incubated at room temperature and wave motion assessed 0.5, 1, 2, 24 and 48 h post supplementation
(prior to assessment, samples were warmed to 37 ◦ C for 2 min). In the absence of seminal plasma or any buffered solution, epididymal wave motion remained consistently high (4.2 ± 0.26) until 48 h post collection when it decreased (1.9 ± 0.69). The addition of tris-citrate-fructose and phosphate buffered saline initially recorded slightly higher wave motion than neat spermatozoa (5.0 ± 0.00 and 4.2 ± 0.59), yet rapidly decreased by 48 h post collection (2.7 ± 0.26 and 0.25 ± 0.10, respectively). Supplementation of epididymal ram spermatozoa with seminal plasma significantly decreased wave motion to the point it had completely ceased by 1 h. This study has shown that exposure to seminal plasma markedly reduced the duration that epididymal spermatozoa exhibited wave motion. Further evidence of this effect is demonstrated by the cessation of wave motion of ejaculated spermatozoa approximately 30 min post collection. Identification of the components within seminal plasma responsible for this effect may be of benefit in extending the life of undiluted ram semen prior to subsequent processing. http://dx.doi.org/10.1016/j.anireprosci.2016.03.057 P39 Plane of nutrition affects scrotal skin thickness and scrotal temperature in pre-pubertal Holstein-Friesian bulls C. Byrne 1,2,∗ , A.M. English 1,3 , S. Fair 3 , P. Lonergan 2 , D.A. Kenny 1,2 1 Animal and Bioscience Department, Teagasc, Dunsany, Co. Meath, Ireland 2 School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland 3 Department of Life Sciences, University of Limerick, Ireland E-mail address: [email protected] (C. Byrne).
The aim of this study was to examine the effect of plane of nutrition on scrotal thermoregulation in pre-pubertal bulls. Autumn-born Holstein-Friesian calves (n = 83) with a mean (±SD) age and bodyweight of 17 (4.4) days and 52 (6.2) kg, respectively, were blocked on age, bodyweight and sire and assigned to a high [H] (n = 37) or low [L] (n = 46) plane of nutrition for the first 6 months of life. At 24 weeks of age, calves either remained on the same, or were switched to the opposite plane of nutrition, until puberty, resulting in four groups: HH; HL; LL and LH. Scrotal skin thickness (SST) was measured monthly (from 16 weeks of age to puberty) using a digital callipers. Thermal images of the scrotum were taken monthly from 28 to 36 weeks of age using a thermal imaging camera. Average scrotal skin surface temperature and gradient (difference in temperature between the testicular vascular cone, TVC and caudal region), were recorded. Data were analysed using the MIXED procedure of SAS (v9.3). Results are presented as mean ± s.e.m. There was a diet × age interaction for SST (P < 0.001). Bulls on H had a greater SST compared to L
was performed using electronic statistics software (SPSS 23.0). Results: A significant relation between sperm motility, concentration and the occurrence of NVs was found. More fast progressive: [(6.2% vs. 1.5%), and slow progressive: (32.0% vs. 19.8%) (p < 0.01) spermatozoa were observed in men with class I sperm. Sperm concentration negatively correlated with MSOME quality (24.1 vs. 6.8 Mio/ml, p < 0.01). No correlation between male age and MSOME quality was observed. Although female partners of men without class I sperm were substantially younger (33.3 vs. 34.3 years, p < 0.001), and the number of retrieved and MII oocytes was higher (12.51 vs. 13.53, p < 0.05; 11.01 vs. 11.76, p < 0.05, respectively), FR, BR and TBR were significant lower when injecting only class II or III sperm: FR: 69.3% vs. 74.9%, p < 0.001; BR: 30.6% vs. 34.9%, p < 0.01; TBR: 10.4% vs. 9.04%, p < 0.05. Conclusions: The negative correlation between conventional semen parameters and occurrence of NV strongly indicates that NVs reflect a pathological situation. This finding is substantiated by a negative impact on early embryonic development. http://dx.doi.org/10.1016/j.anireprosci.2016.03.054 P36 Immunohistochemical localization of paraoxonase type 1 in the boar genital tract Barranco 1 ,
˜ 1, Perez-Patino
Isabel Cristina Alejandro Vicente-Carrillo 2 , Manuel Alvarez-Rodriguez 2 , Emilio A. Martinez 1 , Jordi Roca 1,∗ , Heriberto Rodriguez-Martinez 2 1 Department of Medicine & Animal Surgery, University of Murcia, Spain 2 Department of Clinical & Experimental Medicine, Linköping University, Sweden E-mail address: [email protected] (J. Roca).
The boar seminal plasma (SP) is composed of secretions from testis, epididymis, and accessory sex glands. The enzyme paraoxonase type 1 (PON-1), a high-density lipoprotein-associated enzyme present in boar-SP protects sperm membranes against oxidative damage. Its activity differs among boars, ejaculates within boar and also among ejaculate fractions (Barranco et al., Andrology, 3:315–20, 2015). These findings could indicate different sites of PON-1 expression in the boar genital tract. Accordingly, this study characterized the expression of PON-1 in the boar reproductive tract. Tissue samples from testis, epididymis and accessory sex glands of six healthy and fertile boars slaughtered for gene replacement reasons were fixed in buffered formaldehyde, embedded in paraffin blocks, sliced (4 m thickness), mounted on slides and subjected to PON-1-immunohistochemical localization using a rabbit primary polyclonal antibody against this enzyme (ab53193, Abcam, Cambridge, UK). All boars expressed PON-1 in the genital tract. Within testis, immune reaction
117
against PON-1 was detected in the interstitium (specifically in Leydig cells and blood vessels) and in the seminiferous tubules (specifically in spermatogonia and elongated spermatids). PON-1 markedly stained the cytoplasm of the epididymal principal cells as well as the lumen (secretion and spermatozoa). All accessory sexual glands showed PON-1-staining; the prostatic secretory cells showed a predominantly intracytoplasmic staining leaving the nuclear area negative. The seminal vesicles and bulbourethral glands cells showed a membranous staining. There were no differences in PON-1 localization nor subjective intensity among boars. In conclusion, the present study showed a wide distribution of PON-1 throughout boar genital tract, including testes, epididymis and accessory sexual glands. Supported by MINECO, Madrid, and FEDER Funds, EU (AGL2012-399903); and Seneca Foundation (19892/GERM/15), Murcia, Spain; FORSS and The Swedish Research Councils VR and FORMAS, Stockholm, Sweden. http://dx.doi.org/10.1016/j.anireprosci.2016.03.055 P37 Comparative morphological analysis and optimization of CASA parameters in bull, ram, buck and dog K. Tekin 1,∗ , C. Stelletta 2 , F. Oztutar Stelletta 1 , A. Daskin 1 1 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Ankara University, Turkey 2 Department of Animal Medicine, Production and Health, University of Padova, Italy E-mail address: [email protected] (K. Tekin).
The aim of the study was to optimize the morphological parameters of the CASA system SCA® (Microptic, Barcelona, Spain), comparing with semi-automatic system LAS® (Leica Microsystems, Wetzlar, Germany) in different species. A total of 60 cryopreserved semen samples of four different species (bovine, canine, caprine and ovine) were analysed. Five different semen straws, for each species, from 3 different individuals were used. Smears were fixed for 10 min by adding 1 ml SpermBlue fixative except ram (ram fixed with 3% glutaraldehyde) and stained for 15 min with SpermBlue stain. The slides were analysed with SCA and LAS. Twenty randomly chosen spermatozoa, were evaluated with the morphological module of the SCA and LAS. The sperm head area, acrosome, midpiece and were captured randomly in different fields rejecting only those that overlapped. Area of spermatozoon heads (area, length, with and acrosome), midpieces (area, width and angle) with SCA and heads (length and width), acrosomes (length and width), midpiece length were measured with LAS. To clarify whether species had any ‘differences’ according to staining, one-way ANOVA and Tukey test were performed over the data distribution. Spermatozoon head length and width average of bovine, canine, caprine and ovine were 5.46 ± 0.05, 11.0 ± 0.08; 4.51 ± 0.03, 7.2 ± 0.05;
118
Abstracts / Animal Reproduction Science 169 (2016) 99–135
4.5 ± 0.2, 9.4 ± 0.46; 5.8 ± 0.03, 10.6 ± 0.05 m with SCA; and 9.1 ± 0.06, 4.6 ± 0.04; 5.9 ± 0.04, 3.7 ± 0.03; 8.2 ± 0.05; 4.037 ± 0.04, 8.4 ± 0.03, 4.7 ± 0.04 m with LAS respectively. Angle of insertion midpiece/head shows significant differences (P < 0.05) between different species. Head, acrosome and midpiece parameters from caprine semen were not significantly different with bovine and canine semen. Evaluating the two different methods of analysis, statistically different data have been found in all species (width and length of the head), particularly for bovine, caprine and ovine semen. In order to implement accuracy and repeatability of the SCA® system, improving the algorithm of analysis for ovine and caprine species, as well as increasing the level of standardization throughout the entire process of analysis, appears to be necessary. http://dx.doi.org/10.1016/j.anireprosci.2016.03.056 P38 Exposure to seminal plasma reduces the duration that epididymal ram spermatozoa exhibit wave motion Jessica P. Rickard 1,∗ , Tamara Leahy 1 , Xavier Druart 2 , Simon P. de Graaf 1 1 School of Life and Environmental Sciences, Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia 2 National Institute for Agricultural Research (INRA), University of Tours, 37380 Nouzilly, France E-mail address: [email protected] (J.P. Rickard).
Wave motion (aka mass motility) is commonly used to assess the initial quality of ram semen immediately following ejaculation. However, relatively little is known regarding the mechanisms which govern this phenomenon. One theory suggests that ejaculate sperm concentration plays a role, yet recent work provides evidence to the contrary (David et al., An Reprod Sci, 161, 2015). We hypothesise that contact with seminal plasma at ejaculation influences the initiation and duration of wave motion. As such, this study aims to document epididymal wave motion and observe the influence of seminal plasma on its duration. Epididymal spermatozoa were obtained from the testes of Merino rams (n = 3) at slaughter via microperfusion. Aliquots (25 l) of spermatozoa were then exposed to 50 l of either a tris-citrate-fructose solution, phosphate buffered saline or seminal plasma (previously collected, thawed and warmed as needed). A fourth aliquot of undiluted spermatozoa was used as a control. Wave motion was assessed (6 l on a warmed slide) and scored as per industry standard (1–5; Evans and Maxwell, Artificial insemination of sheep and goats, 1987) immediately post supplementation and every 2 min thereafter until 10 min (samples maintained at 37 ◦ C). Samples were then incubated at room temperature and wave motion assessed 0.5, 1, 2, 24 and 48 h post supplementation
(prior to assessment, samples were warmed to 37 ◦ C for 2 min). In the absence of seminal plasma or any buffered solution, epididymal wave motion remained consistently high (4.2 ± 0.26) until 48 h post collection when it decreased (1.9 ± 0.69). The addition of tris-citrate-fructose and phosphate buffered saline initially recorded slightly higher wave motion than neat spermatozoa (5.0 ± 0.00 and 4.2 ± 0.59), yet rapidly decreased by 48 h post collection (2.7 ± 0.26 and 0.25 ± 0.10, respectively). Supplementation of epididymal ram spermatozoa with seminal plasma significantly decreased wave motion to the point it had completely ceased by 1 h. This study has shown that exposure to seminal plasma markedly reduced the duration that epididymal spermatozoa exhibited wave motion. Further evidence of this effect is demonstrated by the cessation of wave motion of ejaculated spermatozoa approximately 30 min post collection. Identification of the components within seminal plasma responsible for this effect may be of benefit in extending the life of undiluted ram semen prior to subsequent processing. http://dx.doi.org/10.1016/j.anireprosci.2016.03.057 P39 Plane of nutrition affects scrotal skin thickness and scrotal temperature in pre-pubertal Holstein-Friesian bulls C. Byrne 1,2,∗ , A.M. English 1,3 , S. Fair 3 , P. Lonergan 2 , D.A. Kenny 1,2 1 Animal and Bioscience Department, Teagasc, Dunsany, Co. Meath, Ireland 2 School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland 3 Department of Life Sciences, University of Limerick, Ireland E-mail address: [email protected] (C. Byrne).
The aim of this study was to examine the effect of plane of nutrition on scrotal thermoregulation in pre-pubertal bulls. Autumn-born Holstein-Friesian calves (n = 83) with a mean (±SD) age and bodyweight of 17 (4.4) days and 52 (6.2) kg, respectively, were blocked on age, bodyweight and sire and assigned to a high [H] (n = 37) or low [L] (n = 46) plane of nutrition for the first 6 months of life. At 24 weeks of age, calves either remained on the same, or were switched to the opposite plane of nutrition, until puberty, resulting in four groups: HH; HL; LL and LH. Scrotal skin thickness (SST) was measured monthly (from 16 weeks of age to puberty) using a digital callipers. Thermal images of the scrotum were taken monthly from 28 to 36 weeks of age using a thermal imaging camera. Average scrotal skin surface temperature and gradient (difference in temperature between the testicular vascular cone, TVC and caudal region), were recorded. Data were analysed using the MIXED procedure of SAS (v9.3). Results are presented as mean ± s.e.m. There was a diet × age interaction for SST (P < 0.001). Bulls on H had a greater SST compared to L