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British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
‘Serum like’ albumin of fowl seminal plasma and effects of albumin on fowl spermatozoa stored at 4°c a
E. Blesbois & J. P. Caffin
a
a
INRA Station de Recherches Avicoles , 37380, Nouzilly, France Published online: 08 Nov 2007.
To cite this article: E. Blesbois & J. P. Caffin (1992) ‘Serum like’ albumin of fowl seminal plasma and effects of albumin on fowl spermatozoa stored at 4°c, British Poultry Science, 33:3, 663-670, DOI: 10.1080/00071669208417504 To link to this article: http://dx.doi.org/10.1080/00071669208417504
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/ page/terms-and-conditions
British Poultry Science (1992) 33: 663-670
'SERUM LIKE' ALBUMIN OF FOWL SEMINAL PLASMA AND EFFECTS OF ALBUMIN ON FOWL SPERMATOZOA STORED AT 4°C E. BLESBOIS AND J. P. CAFFIN INRA Station de Recherches Avicoles, 37380 Nouzilly, France
Downloaded by [] at 02:32 30 December 2014
Received for publication 12th July 1991
Abstract 1. Using immunoelectrophoretic and immunodiffusion methods, serum-like albumin was detected in fowl seminal plasma. Immunodiffusion showed seminal plasma albumin concentration to be 4 mg/ml, corresponding to half of the total proteins (8 mg/ml). 2. Replacing seminal plasma with a diluent containing either 1, 4, or 16 mg/ml albumin increased motility of spermatozoa stored for 24 h at 4°C, 16 mg/ml being the more effective dose. 1 and 4 mg/ml had no effect on the fertilising ability of fowl spermatozoa stored for 24 h at 4°C in both young (28-35 weeks old) and old birds (50-55 weeks old). 16 mg/ml albumin had no effect on fertilising rates in young but depressed it in old birds. 3. These results indicate that seminal plasma albumin may be one of the mobility stimulating factors of seminal plasma. However it does not protect fertilising ability better than the diluent alone. INTRODUCTION
In domestic fowl, seminal plasma stimulates the motility (Terada et al., 1978) and the fertilising ability (Lake and Ravie, 1987) of fresh sperm but damages the fertilising ability of spermatozoa stored in vitro (Blesbois and Mauger, 1987; Sexton, 1988). The lower molecular weight fraction of seminal plasma ( < 1,000 dalton) is responsible for the deleterious effect of seminal plasma on stored spermatozoa (Blesbois and de Reviers, 1992). The highest molecular weight (hmw) fraction has no such toxic effect and seems to play a favourable role in sperm storage. The hmw fraction of seminal plasma contains numerous proteins which have been poorly described. According to Kichev and Danov (1972) who suggested the presence of a serum like albumin in seminal plasma of fowl, PAGE electrophoresis of the hmw fraction of seminal plasma shows that a seminal protein migrates at the same rate as chicken blood serum albumin (CSA). The aim of the present study is to confirm the presence and role of the serum-like albumin of fowl seminal plasma: is serum-like albumin really present in fowl seminal plasma and is it immunologically identical to blood serum albumin? If so, what is the ratio of serum albumin to total protein in seminal 663
664
E. BLESBOIS AND J. P. CAFFIN
plasma? If so, do biological concentrations of CSA influence sperm motility and fertilising ability in fowls? MATERIALS AMD METHODS
Animals
The 48 heavy male birds were of the Cornish strain 199. The females were 500 dwarf 'Vedette' JV 15 broiler breeders. At the time of the study, all birds were between 30 and 55 weeks of age and were housed in individual battery cages. Food containing 11 -7 MJ of ME and 150 g of protein/kg was rationed at 100 g/d for males and 120 g/d for females.
Downloaded by [] at 02:32 30 December 2014
Semen collection
Semen was collected by manual massage (Burrows and Quinn, 1937), care being taken to avoid contamination of semen with cloacal products and transparent fluid excreted from the lymph folds of the cloaca during ejaculation. Preparation of seminal plasma
For the purposes of the determination of the serum albumin content of seminal plasma, mixed ejaculates from 25 males were used. Seminal plasma was separated from spermatozoa by three successive centrifugations. The first centrifugation (500 Xg for 10 min. at 20°C) of whole semen was performed no more than 10 min. after the start of the semen collection session. The following two centrifugations of the supernatants were carried out at 4°C, respectively at 500 Xg for 10 min. and 3000 Xg- for 30 min. After extraction, seminal plasma was frozen and stored for subsequent analysis. The content of protein of seminal plasma was measured by the method of Lowry et al. (1951). Characterisation and determination of chicken serum-like albumin
Chicken serum like albumin (CSA) was detected in seminal plasma by the double immunodiffusion technique of Ouchterlony (1948) and by immunoelectrophoresis (Scheidegger, 1955) using specific rabbit antibodies to chicken whole blood serum, immunoglobulins, ovalbumin or serum albumin (Nordic Immunological Laboratories, N.L.). The plates were prepared with agarose (Difco) (15 g/1) in 0-05 M veronal buffer (pH 8-6). The concentration of chicken serum albumin (CSA, see Results) in seminal plasma was measured by the single radial immunodiffusion method of Mancini et al. (1965). The plates prepared as above, contained 20 [A of specific rabbit antibodies to CSA. Standard solutions of CSA (1, 2, 3, 4, 5 mg/ml) for each plate were made up in 0-14 M NaCl-0-01 M phosphate buffer, pH 7-2. To estimate the concentration of CSA, 5 fll of samples or standard solutions were applied to the wells of gelose plates with a Hamilton syringe. Each sample was
SERUM ALBUMIN AND STORAGE OF FOWL SPERM
665
analysed twice. After a 48 h incubation at 20°C, precipitin rings were washed and dried, stained with Coomassie Brilliant Blue R 250 (Sigma), and their diameters measured. Linear regression analysis was used to establish a calibration curve for each plate by plotting the square root of the concentration of CSA standard solutions against diameters of precipitin rings and CSA concentration of unknown samples was calculated. Storage of sperm
Downloaded by [] at 02:32 30 December 2014
For measurements of motility and fertilising ability, semen from 10 to 12 birds was pooled and the concentration of spermatozoa estimated immediately using a photometer set at a wavelength of 540 nm. Samples were then divided into 1 ml fractions. Within experiments and treatment groups, pooled semen was used. For the storage of whole diluted semen, semen was diluted 1:1 in Beltsville Poultry Semen Extender (BPSE, Sexton, 1977) and stored for 24 h at 4°C. For the storage of spermatozoa without seminal plasma, samples were centrifuged for 10 min at 500 Xg (Sexton, 1973). The resulting supernatant was discarded and replaced by BPSE alone or by BPSE containing 1, 4 or 16 mg/ml albumin. Because albumin of seminal plasma was immunologically identical to blood serum albumin (see Results), chicken blood serum albumin was purchased from Nordic Immunological Laboratories, N.L. pH was adjusted to 7-2 and osmolarity to 350 mosm. This reconstituted semen was then diluted 1:1 in BPSE diluent and stored for 24 h at 4°C and in aerobic conditions (400 nM O2/ml). Motility and fertilising ability of sperm
Motility was observed under a light microscope and subjectively noted on a scale ranging from 0 to 10 (Petitjean, 1965). Each sample was measured twice and 7 replicates were examined for each treatment. Samples were examined between slides and coverslips which were either untreated or pretreated with Repel Silane (LKB Produkter A.B., Bromma, Sweden) which avoids the adhesion of the cells to the glass. There was no difference in the motility of the same samples with treated or untreated glass and only results with untreated glasses are reported here. The fertilising ability of the spermatozoa was estimated from the results of intravaginal inseminations of 200 million spermatozoa/female/ week over 4 consecutive weeks. 90 females were inseminated per treatment. Eggs were collected on days 2 to 8 inclusive after each insemination. Fertilisation rates (percentage of fertilised eggs/incubated eggs) were determined by candling the eggs 7 d after the start of incubation. The rates of hatchability (percentage of hatched eggs/fertilised eggs) were determined after 21 d of incubation. The motility data were examined by analysis of variance and the NewmanKeuls tests. For fertilisation rates, heterogeneity of variance and non-normal distribution (even following arc sin Vx, cos x, log x and logit transformations) made classical analysis of variance inappropriate (Dagnelie, 1975), and Pearson X2 test was used.
666
E. BLESBOIS AND J. P. CAFFIN 7-5 -,
7-0-
= 6-5-
Downloaded by [] at 02:32 30 December 2014
6-0-
5-5 J 24
Hours of storage
FIG—Effect of replacement of seminal plasma by a diluent containing 0, 1, 4 or 16 mg/ml albumin on the motility of spermatozoa stored for 24 h at 4°C. The standard fresh unstored semen is represented by D. All other samples with or without seminal plasma (SP) were stored 24 h diluted 1:1 in BPSE: (A—A) whole semen, (A A) SP replaced by BPSE, (• •) SP replaced by BPSE plus 1 mg/ml albumin, (O O) SP replaced by BPSE plus 4 mg/ml albumin, (• •) SP replaced by BPSE plus 16 mg/ml albumin. Points bearing different letters differ significantly (P
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
‘Serum like’ albumin of fowl seminal plasma and effects of albumin on fowl spermatozoa stored at 4°c a
E. Blesbois & J. P. Caffin
a
a
INRA Station de Recherches Avicoles , 37380, Nouzilly, France Published online: 08 Nov 2007.
To cite this article: E. Blesbois & J. P. Caffin (1992) ‘Serum like’ albumin of fowl seminal plasma and effects of albumin on fowl spermatozoa stored at 4°c, British Poultry Science, 33:3, 663-670, DOI: 10.1080/00071669208417504 To link to this article: http://dx.doi.org/10.1080/00071669208417504
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/ page/terms-and-conditions
British Poultry Science (1992) 33: 663-670
'SERUM LIKE' ALBUMIN OF FOWL SEMINAL PLASMA AND EFFECTS OF ALBUMIN ON FOWL SPERMATOZOA STORED AT 4°C E. BLESBOIS AND J. P. CAFFIN INRA Station de Recherches Avicoles, 37380 Nouzilly, France
Downloaded by [] at 02:32 30 December 2014
Received for publication 12th July 1991
Abstract 1. Using immunoelectrophoretic and immunodiffusion methods, serum-like albumin was detected in fowl seminal plasma. Immunodiffusion showed seminal plasma albumin concentration to be 4 mg/ml, corresponding to half of the total proteins (8 mg/ml). 2. Replacing seminal plasma with a diluent containing either 1, 4, or 16 mg/ml albumin increased motility of spermatozoa stored for 24 h at 4°C, 16 mg/ml being the more effective dose. 1 and 4 mg/ml had no effect on the fertilising ability of fowl spermatozoa stored for 24 h at 4°C in both young (28-35 weeks old) and old birds (50-55 weeks old). 16 mg/ml albumin had no effect on fertilising rates in young but depressed it in old birds. 3. These results indicate that seminal plasma albumin may be one of the mobility stimulating factors of seminal plasma. However it does not protect fertilising ability better than the diluent alone. INTRODUCTION
In domestic fowl, seminal plasma stimulates the motility (Terada et al., 1978) and the fertilising ability (Lake and Ravie, 1987) of fresh sperm but damages the fertilising ability of spermatozoa stored in vitro (Blesbois and Mauger, 1987; Sexton, 1988). The lower molecular weight fraction of seminal plasma ( < 1,000 dalton) is responsible for the deleterious effect of seminal plasma on stored spermatozoa (Blesbois and de Reviers, 1992). The highest molecular weight (hmw) fraction has no such toxic effect and seems to play a favourable role in sperm storage. The hmw fraction of seminal plasma contains numerous proteins which have been poorly described. According to Kichev and Danov (1972) who suggested the presence of a serum like albumin in seminal plasma of fowl, PAGE electrophoresis of the hmw fraction of seminal plasma shows that a seminal protein migrates at the same rate as chicken blood serum albumin (CSA). The aim of the present study is to confirm the presence and role of the serum-like albumin of fowl seminal plasma: is serum-like albumin really present in fowl seminal plasma and is it immunologically identical to blood serum albumin? If so, what is the ratio of serum albumin to total protein in seminal 663
664
E. BLESBOIS AND J. P. CAFFIN
plasma? If so, do biological concentrations of CSA influence sperm motility and fertilising ability in fowls? MATERIALS AMD METHODS
Animals
The 48 heavy male birds were of the Cornish strain 199. The females were 500 dwarf 'Vedette' JV 15 broiler breeders. At the time of the study, all birds were between 30 and 55 weeks of age and were housed in individual battery cages. Food containing 11 -7 MJ of ME and 150 g of protein/kg was rationed at 100 g/d for males and 120 g/d for females.
Downloaded by [] at 02:32 30 December 2014
Semen collection
Semen was collected by manual massage (Burrows and Quinn, 1937), care being taken to avoid contamination of semen with cloacal products and transparent fluid excreted from the lymph folds of the cloaca during ejaculation. Preparation of seminal plasma
For the purposes of the determination of the serum albumin content of seminal plasma, mixed ejaculates from 25 males were used. Seminal plasma was separated from spermatozoa by three successive centrifugations. The first centrifugation (500 Xg for 10 min. at 20°C) of whole semen was performed no more than 10 min. after the start of the semen collection session. The following two centrifugations of the supernatants were carried out at 4°C, respectively at 500 Xg for 10 min. and 3000 Xg- for 30 min. After extraction, seminal plasma was frozen and stored for subsequent analysis. The content of protein of seminal plasma was measured by the method of Lowry et al. (1951). Characterisation and determination of chicken serum-like albumin
Chicken serum like albumin (CSA) was detected in seminal plasma by the double immunodiffusion technique of Ouchterlony (1948) and by immunoelectrophoresis (Scheidegger, 1955) using specific rabbit antibodies to chicken whole blood serum, immunoglobulins, ovalbumin or serum albumin (Nordic Immunological Laboratories, N.L.). The plates were prepared with agarose (Difco) (15 g/1) in 0-05 M veronal buffer (pH 8-6). The concentration of chicken serum albumin (CSA, see Results) in seminal plasma was measured by the single radial immunodiffusion method of Mancini et al. (1965). The plates prepared as above, contained 20 [A of specific rabbit antibodies to CSA. Standard solutions of CSA (1, 2, 3, 4, 5 mg/ml) for each plate were made up in 0-14 M NaCl-0-01 M phosphate buffer, pH 7-2. To estimate the concentration of CSA, 5 fll of samples or standard solutions were applied to the wells of gelose plates with a Hamilton syringe. Each sample was
SERUM ALBUMIN AND STORAGE OF FOWL SPERM
665
analysed twice. After a 48 h incubation at 20°C, precipitin rings were washed and dried, stained with Coomassie Brilliant Blue R 250 (Sigma), and their diameters measured. Linear regression analysis was used to establish a calibration curve for each plate by plotting the square root of the concentration of CSA standard solutions against diameters of precipitin rings and CSA concentration of unknown samples was calculated. Storage of sperm
Downloaded by [] at 02:32 30 December 2014
For measurements of motility and fertilising ability, semen from 10 to 12 birds was pooled and the concentration of spermatozoa estimated immediately using a photometer set at a wavelength of 540 nm. Samples were then divided into 1 ml fractions. Within experiments and treatment groups, pooled semen was used. For the storage of whole diluted semen, semen was diluted 1:1 in Beltsville Poultry Semen Extender (BPSE, Sexton, 1977) and stored for 24 h at 4°C. For the storage of spermatozoa without seminal plasma, samples were centrifuged for 10 min at 500 Xg (Sexton, 1973). The resulting supernatant was discarded and replaced by BPSE alone or by BPSE containing 1, 4 or 16 mg/ml albumin. Because albumin of seminal plasma was immunologically identical to blood serum albumin (see Results), chicken blood serum albumin was purchased from Nordic Immunological Laboratories, N.L. pH was adjusted to 7-2 and osmolarity to 350 mosm. This reconstituted semen was then diluted 1:1 in BPSE diluent and stored for 24 h at 4°C and in aerobic conditions (400 nM O2/ml). Motility and fertilising ability of sperm
Motility was observed under a light microscope and subjectively noted on a scale ranging from 0 to 10 (Petitjean, 1965). Each sample was measured twice and 7 replicates were examined for each treatment. Samples were examined between slides and coverslips which were either untreated or pretreated with Repel Silane (LKB Produkter A.B., Bromma, Sweden) which avoids the adhesion of the cells to the glass. There was no difference in the motility of the same samples with treated or untreated glass and only results with untreated glasses are reported here. The fertilising ability of the spermatozoa was estimated from the results of intravaginal inseminations of 200 million spermatozoa/female/ week over 4 consecutive weeks. 90 females were inseminated per treatment. Eggs were collected on days 2 to 8 inclusive after each insemination. Fertilisation rates (percentage of fertilised eggs/incubated eggs) were determined by candling the eggs 7 d after the start of incubation. The rates of hatchability (percentage of hatched eggs/fertilised eggs) were determined after 21 d of incubation. The motility data were examined by analysis of variance and the NewmanKeuls tests. For fertilisation rates, heterogeneity of variance and non-normal distribution (even following arc sin Vx, cos x, log x and logit transformations) made classical analysis of variance inappropriate (Dagnelie, 1975), and Pearson X2 test was used.
666
E. BLESBOIS AND J. P. CAFFIN 7-5 -,
7-0-
= 6-5-
Downloaded by [] at 02:32 30 December 2014
6-0-
5-5 J 24
Hours of storage
FIG—Effect of replacement of seminal plasma by a diluent containing 0, 1, 4 or 16 mg/ml albumin on the motility of spermatozoa stored for 24 h at 4°C. The standard fresh unstored semen is represented by D. All other samples with or without seminal plasma (SP) were stored 24 h diluted 1:1 in BPSE: (A—A) whole semen, (A A) SP replaced by BPSE, (• •) SP replaced by BPSE plus 1 mg/ml albumin, (O O) SP replaced by BPSE plus 4 mg/ml albumin, (• •) SP replaced by BPSE plus 16 mg/ml albumin. Points bearing different letters differ significantly (P
