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Elevated Seminal Plasma Protein: A Characteristic of Yellow Turkey Semen1
PHYSIOLOGY AND REPRODUCTION Elevated Seminal Plasma Protein: A Characteristic of Yellow Turkey Semen1 R. J. THURSTON, R. A. HESS, and D. P. FROMAN Department of Poultry Science, Clemson University, Clemson, South Carolina 29631 H. V. BIELLIER Department of Poultry Husbandry, University of Missouri, Columbia, Missouri 65211 (Received for publication December 1, 1981)
1982 Poultry Science 61:1905-1911
INTRODUCTION
Yellow turkey semen contains abnormal spermatozoa, spermatids, often numerous macrophages, and has reduced fertilizing capability when compared to normal white semen (Saeki and Brown, 1962; Cherms, 1968; Thurston and Biellier, 1972; Marquez and Ogasawara, 1975; Thurston et ai, 1975). However, the biochemical characteristics of yellow semen are unknown. Preliminary experiments designed to identify the yellow substance showed an elevated concentration of seminal plasma proteins (unpublished data). This finding provided die impetus for the research reported herein. The objectives were 1) to monitor semen quality weekly for 15 weeks (i.e., seminal plasma color, protein concentration and subfractions) and semen volume of males that initially produced white (WS) or
1 Published with approval of die director of the South Carolina Agricultural Experiment Station as Technical Contribution No. 1994.
yellow (YS) semen and 2) to determine the correlation between blood and seminal plasma protein concentration, and 3) to compare the mobility and staining pattern of blood and seminal plasma proteins separated by electrophoresis. MATERIALS AND METHODS
Weekly Evaluation of White Versus Yellow Semen. Large White turkey breeder males (60) were stimulated to produce semen in December with incandescent light (12 lx, 14L:10D). Semen was collected for 5 weeks and evaluated as white or yellow by visual inspection. Eight males producing WS and 8 YS were selected for a further 15-week study of semen quality. All toms were 3 3 weeks old with the exception of two YS males that were approximately 2 years old. The older males were included as Hess (personal communication) found no effect on yellow semen production or characterstics due to age. The males were randomly housed in 1.83 X 2.44 m pens, 4 per pen, and fed a
1905
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ABSTRACT Semen volume, seminal plasma color, protein concentration, and subfractions from 8 white semen (WS) and 8 yellow semen (YS) Large White turkeys were measured for 15 weeks. Average volume was .29 ± .01 ml for WS and .32 ± .02 ml for YS males. Plasma from WS was white and averaged 1.84 ± .07 g/100 ml protein, whereas YS seminal plasma was yellow with 7.03 ± .5 g/100 ml protein. Protein subfractions in WS and YS seminal plasma were qualitatively similar and consisted of prealbumin, albumin, alpha—1 and —2, beta—1, —2, and —3, and postbeta (gamma). The alpha proteins were more frequently seen in YS seminal plasma. The elevated protein of YS seminal plasma was due to an increased concentration of all proteins, with the possible exception of beta—3, with albumin contributing the most. Except for prealbumin and beta—3, the seminal proteins had the same electrophoretic mobility as counterpart blood proteins. Blood and seminal plasma protein concentrations were poorly correlated (+ .039; P<.75). Blood protein concentrations from 68 toms ranged from 3.3 to 5.7 g/100 ml and averaged 4.2 g/100 ml; seminal plasma protein concentrations ranged from .93 to 12.8 g/100 ml. Nineteen percent of the males had YS with seminal plasma protein greater than 4.0 g/100 ml. The high correlation between intensity of the yellow color in YS seminal plasma and protein concentration (+ .76; P<.01) indicates that semen quality of turkey breeders could be improved by selection on the basis of low seminal plasma protein concentration. (Key words.- turkey, blood, semen, plasma, proteins)
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THURSTON ET AL.
The protein concentration of frozen-thawed seminal plasma was determined by the biuret method as described by Thurston (1976). A standard curve was prepared using 1 to 16 g/100 ml of chicken blood plasma albumin (Sigma, St. Louis, MO), and a least squares equation of optical density versus albumin concentration was used to determine seminal plasma protein concentration. Only one de-
termination could be made per sample due to the small volume of seminal plasma. Comparison of Seminal and Blood Plasma Proteins. Heparinized (10 units/ml) brachial vein blood and semen were collected from 68 Large White breeder males (42 weeks of age). Plasma was collected by centrifugation and protein concentration was determined by the biuret method, with two measurements per sample. Blood and seminal plasma from 4 WS and 4 YS males were chosen for comparison of plasma protein fractions by electrophoresis; 8 /il of blood plasma were applied per gel. Statistical Analysis. Statistical analysis was with a nested arrangement for analysis of variance, mean differences determined by the method of least significance difference. A model for semen volume by week was determined by the method of least squares. RESULTS
Comparison of Semen Quality of White and Yellow Semen Males. Average semen volume was greater for YS males (P<.05), .32 ± .02 ml versus .29 ± .01 ml for the WS males. The best model for semen volume for the 15 weeks was parallel lines (not rejected at P<.05) with negative slopes (-.006 for YS; - . 0 0 2 for the WS males), indicating that volume slightly declined for both groups as the season progressed. Semen volume of YS toms was greater than for WS males except for 2 weeks. The average seminal plasma color of the YS males was yellow to dark yellow (average code = 3.5 ± .14; Table 1) compared to white (average code = 1.3 ± .10) for WS males. The correlation between degree of yellow color and seminal plasma protein concentration was high + .76 (P«.01), due to increased protein in YS seminal plasma, which averaged 7.03 ± .5 g/100 ml versus 1.84 ± .07 for WS males (P«.05; Table 1). Seminal plasma protein concentration showed little variation by week in the WS males but was more variable for YS males (Fig. 1). This was partially due to grouping on the basis of semen color. The plasma begins to appear yellow when the protein concentration is approximately 2.5 to 3.0 g/100 ml. Thus, males with white semen would have seminal plasma protein concentrations confined to a narrow range (average range was 1.38 to 3.0 g/100 ml), whereas a male classified as YS had an excess of 2.5 to 3.0 g/100 ml, but there was no limit on
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turkey breeder ration (16.6% protein; 2641 kcal ME/kg) ad lib. Semen was collected by massage (Burrows and Quinn, 1937) into tuberculin syringes, and volume was recorded. The semen was placed into 5 ml centrifuge tubes and centrifuged at 2000 X g at 4 C for 30 min. Seminal plasma color was estimated (against a light blue background) as white, slight yellow, yellow, or dark yellow and coded 1, 2, 3, or 4, respectively. The plasma was removed and stored in 1 ml vials at 4 C for electrophoresis the following day. Plasma proteins were separated using polyacrylamide disc gel electrophoresis. Gels were polymerized in 11.4 X .5 (i.d.) cm glass tubes, an 8.4 cm, 7% acrylamide separating gel adjusted to pH 8.9 with a Tham-HCl (Fisher Scientific Co., St. Louis, MO) buffer, overlayered with a 2.4 cm, 4% acrylamide stacking gel at pH 6.7 (Tham-HCl buffer). Polymerization of the gel solutions was achieved by adding .01 g/100 ml ammonium persulfate. A Thamglycine buffer, pH 8.3, was used in the cathode and anode chambers. Ten microliters of white or 4 fil of yellow seminal plasma were injected on top of the stacking gel with a microsyringe (except for YS males with <2.5 g/100 ml of protein where 10 /il were added). The remaining plasma was frozen. Electrophoresis was performed using constant voltage; 100 V for 1 hr, then 280 V for 1.5 hr. The gels were stained with amido black for 10 min, electrically destained, cleared with 5% glycerol, and scanned with a Densicord Model 542 densitometer (Photovolt Corp., New York, NY) using visible light. Peak area was integrated as densitometer units by a Photovolt Model 49A integrator. Electrophoresis and densitometer scan of purified turkey seminal plasma albumin showed that peak area (densitometer units) was linearly related to protein concentration between 10 and 230 /Ug of protein (r2 = .96). Therefore, peak area approximated concentration and was used for quantitation of individual protein fractions.
ELEVATED PROTEIN IN YELLOW TURKEY SEMEN
the maximum seminal protein concentration, which was as high as 13.65 g/100 ml. The large variation in seminal plasma protein by week was also due to within male variation. For example, for two males seminal plasma protein declined-, from 4.4 g/100 ml on week 1 to 1.3 g/100 ml at week 15 for one (Fig. 2), and from 10.5 g/100 ml at week 6 to 3.1 g/100 ml at week 15 for the other. Accompanying the diminution in protein was a change in plasma color from yellow to white and slight yellow. None of the WS males changed to chronic YS producers, aldiough two occasionally produced semen with yellow colored plasma and elevated protein. Six of the YS males had elevated seminal plasma protein concentrations for the entire 15 weeks concomitant with yellow or dark yellow plasma, but their protein level varied by week. The most pronounced example of the YS syndrome was exhibited by a male whose protein ranged from 9.4 to 19.5 g/100 ml, and the plasma color was always dark yellow. In contrast, a constant WS male had white plasma whose protein ranged from 1.1 to 1.9 g/100 ml for the 15-week period (Fig. 2). Six major protein fractions were consistently separated from white seminal plasma (Fig. 3). They were identified on the basis of mobility using the nomenclature of Bierer (1969) for turkey serum proteins. In order, beginning with those which migrated farthest from the cathode, were: prealbumin, albumin, beta—1, —2, and —3, and postbeta (gamma) proteins. Alpha—1 and —2 proteins were occasionally detected at low concentrations. Albumin was the most abundant protein, representing 65 to
70% in 6 males, but in the remaining 2 toms with WS it was 50% and 34%. For the latter males, the beta—2 and —3 fractions represented a greater percentage of total protein. The proteins in YS plasma were qualitatively similar to those of WS plasma, but alpha—1, and especially alpha—2, were more frequently observed, particularly in males that had the highest level of seminal plasma protein (Fig. 4). The elevated protein of YS plasma was due to increased concentrations of prealbumin (P<.01), Albumin (P< .01), alpha-2 (P«.05), b e t a - 1 (P<.05), b e t a - 2 (P«.05), and postbeta (P<.05) proteins (Table 1). The average beta—3 peak area for YS (58.4; Table 1) was larger than for WS (45.3; Table 1), but this difference was not statistically significant at (P«.05). Thus all proteins, with the possible exception of beta—3, contributed to the elevated protein in YS seminal plasma. There was a disproportionate contribution to the elevated seminal plasma protein by each of the increased protein fractions in YS (Table 2). The average percent albumin of total seminal protein in six YS males, 81.4%, was 20.3% greater than for WS males, and this is
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FIG. 2. Seminal plasma protein concentration by week of a WS male and two YS males. The protein concentration of the WS male (o) represents the normal pattern, i.e., between 1 and 2 g/100 ml. One YS male (a) recovered from the YS syndrome (< protein), whereas the other (A) was a chronic YS producer.
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FIG. 1. Average seminal plasma protein concentration by week ± SEM. The elevated seminal plasma protein concentration of YS males is evident.
1907
1908
THURSTON ET AL.
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FIG. 3. Gel and densitometer scan of proteins resolved from 10 jul of normal white seminal plasma. 1 = prealbumin; 2 = albumin; 3 = beta—1; 4 = beta—2; 5 = beta— 3; 6 = postbeta (gamma).
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probably a conservative estimate, because the albumin b a n d in YS seminal plasma stained so intensely that t h e d e n s i t o m e t e r response was nonlinear. T h u s , albumin contributes m o s t to t h e increased plasma p r o t e i n in YS. For the other t w o YS t o m s , albumin was 61.6 and 74.4% of total protein, respectively, even
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FIG. 4. Gel and densitometer scan of proteins resolved from 4.0 )j.\ of yellow seminal plasma. Although 2.5 times less plasma was used than for white plasma (Fig. 3). the proteins are more concentrated, and two alpha fractions are resolved. 1 = prealbumin; 2 = albumin; 3 = alpha-1; 4 = alpha—2; 5 = b e t a - 1 ; 6 = beta—2; 7 = beta—3; 8 = postbeta (gamma).
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ELEVATED PROTEIN IN YELLOW TURKEY SEMEN
1909
DISCUSSION Turkey males producing YS have welldeveloped secondary sexual characteristics and
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FIG. 5. Gels depicting blood (top gel) and seminal plasma proteins from a YS male. The scan represents the fractions in blood plasma. 1 = prealbumin; 2 • albumin; 3 = alpha—1; 4 = alpha—2; 5 = beta—1; 6 = beta—2; 7 = b e t a - 3 ; 8 = postbeta (gamma).
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though much more was present than in the seminal plasma of the WS males. This was mainly due to an elevated postbeta fraction, which contributed a greater amount to the total protein than for other YS males. Comparison of Blood with Seminal Plasma Proteins. There was low correlation (+ .039; P<.75) between blood and seminal plasma protein concentrations, which ranged from 3.3 to 5.7 g/100 ml with an average of 4.2 ± .04 g/100 ml for blood and ranged from .93 to 12.8 g/100 ml for seminal plasma. Thirteen of the 68 males (19%) had yellow seminal plasma with protein greater than 4.0 g/100 ml. Eight protein fractions were consistently separated from blood plasma. In order of farthest migration from die cathode they were: prealbumin, albumin, alpha—1 and —2, beta—1, —2, and —3, and postbeta (gamma). For both YS and WS males, the seminal plasma albumin, alpha—1 and —2, and beta—1 and —2 protein fractions generally had the same mobility as corresponding proteins in blood plasma (Fig. 5). Seminal plasma beta—3 migrated differently than the beta—3 protein of blood plasma.
1910
THURSTON ET AL.
The YS was characterized by elevated seminal plasma protein, the concentration highly correlated with the degree of yellow plasma color. It is interesting that Crabo et al. (1971) found that the plasma removed from the epididymis of a bull with abnormal sper-
matozoa also had a higher protein concentration than normal. The yellow color is confined to the plasma (Cherms, 1968), and precipitation of the plasma proteins removes the yellow color, so it is possible that the yellow discoloration is due to the excess protein. The elevated protein was caused by an increased concentration of proteins found 'in normal WS seminal plasma, with the exception of the beta—3. Because most of the elevated YS seminal plasma proteins were similar to blood plasma proteins, blood proteins could possibly get into the seminal fluids and contribute to the increased seminal protein. Alternatively, the protein may be synthesized and released by reproductive tissue. Hess et al. (1982) found that the testes and epididymal region of YS males were yellow. Microscopy revealed that the ductuli efferentes epithelia were hypertrophied and filled with lipid-like vacuoles, and many cells appeared to bleb into the lumen. Lin and Chang (1975) showed that albumin is synthesized by a variety of epithelial types; hence, the excess protein in YS may come from the abnormal efferentes epithelia. Albumin/beta and albumin/postbeta ratios for YS seminal plasma were larger (6 of 8 toms) than for WS, indicating that albumin was disproportionally greater in YS than other proteins. Thus, the elevated protein concentration of YS was not achieved by removal of water, which would concentrate all proteins proportionately. Yellow semen is known to contain abnormal sperm cells and spermatids (Saeki and Brown, 1962; Marquez and Ogasawara, 1975; Thurston et al, 1975), so quantitation of abnormal cells was parenthetical to the objectives of the present research. However, each sample was briefly scored for abnormal cells by microscopic inspection. The number of abnormal cells declined simultaneously with the change in plasma color and decline in seminal plasma protein in the two YS males with transient YS production. These observations, in addition to the fact that the seminal plasma protein of chronic YS males oscillated over time, is evidence that the degree of involvement with the YS syndrome is dynamic. Because of their poor fertilizing capability, current management practices recommend that YS males be culled from breeder flocks. The problem is recognition of the yellow color in whole semen. One can visually detect yellow in the plasma when the protein is approximately 2.5 to 3.0 g/100 ml.
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testes size comparable to those of WS males (Thurston, 1976). These observations and the finding that average semen volume of YS males was slightly more than for WS males for 15 weeks is evidence that the YS syndrome is not the result of refractoriness. The severity of the YS syndrome (degree of seminal plasma protein elevation) was not negatively correlated with semen volume, but the YS male with die most plasma protein (average 13.65 g/100 ml) gave the least volume (average .21 ml) of all YS males. Further research is needed to determine if there is a relationship between semen volume and the degree of plasma protein elevation. The average seminal plasma protein of WS males, 1.84 g/100 ml, was lower than the 2.7 g/100 ml reported by Graham et al. (1971), whose data was obtained from a flock that probably contained YS males. Only 6 major proteins were separated from WS seminal plasma, whereas 14 have been identified in chicken and 9 in guinea seminal plasma (Harris and Sweeney, 1971; Thurston et al, 1982). Based on staining profile and electrophoretic mobility, the turkey seminal plasma proteins, except prealbumin and beta—3, were similar to proteins separated from blood plasma. As for blood, albumin was the most abundant protein, but between male variation in quantity was noted, a possible reflection of different genotypes, as the concentration and phenotype of blood albumin in chickens and chicken-turkey hybrids has been shown to be governed by inherited traits (Fried and Chun, 1971; Phillips et al, 1973). Seminal plasma beta proteins may be transferrin, because their relative mobility and staining pattern were similar to beta—1 and —2 chicken blood plasma transferrins (Glick, 1968; Torres-Medina et al, 1971). B e t a - 3 , the most abundant seminal beta protein, was closely associated with the beta—1 and —2 and may represent transferrin synthesized by cells of the reproductive tract. Similarly, O'Rand (1974) characterized a lactoferrin-like protein in rabbit seminal plasma that differed from blood transferrins. The postbeta fraction was diffuse in WS seminal plasma and probably represents a conglomerate of gamma globulins, possibly antibodies.
ELEVATED PROTEIN IN YELLOW TURKEY SEMEN Plasma protein concentration usually must exceed 4 to 5 g/100 ml before whole semen can be detected as yellow, but acuity is not good in low intensity lighting regimens. Thus, culling of YS males based on visualization of whole semen is subjective with large error. Screening for elevated seminal plasma protein should improve the semen quality of breeder turkey males and help identify semen problems in flocks experiencing idiopathic infertility.
REFERENCES Bierer, B. W., 1969. Serum protein fractions in the normal turkey. Poultry Sci. 48:1208-1215. Burrows, W. H., and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16:19-24. Cherms, F., 1968. Variations in semen quality and the relationship of semen quality to fertility in turkeys. Poultry Sci. 47:746-754. Crabo, B., L. Gustafsson, and A. R. Rao, 1971. Subnormal testicular function in a bull concealed by phagocytosis of abnormal spermatozoa in the efferent ductules. J. Reprod. Fert. 26:393-396. Fried, M., and P. W. Chun, 1971. Polymorphism in fowl serum albumin-I. Characterization of two genetic variants of fowl serum albumin and definition of peptide differences. Comp. Bio-
chem. Physiol. 39B:523-540. Glick, B., 1968. Serum protein electrophoretic patterns in acrylamide gel: patterns from normal and bursaless birds. Poultry Sci. 47:807-814. Graham, E. F., M. L. Schmehl, K. I. Brown, B. G. Crabo, and G. Ertel, 1971. Some chemical studies of the female reproductive tract and seminal plasma of the male turkey and their relationship to fertility. Poultry Sci. 50:1170-1181. Harris, G. C. Jr., and M. J. Sweeney, 1971. Changes in the protein concentration of chicken seminal plasma after rapid freeze-thaw. Cryobiology 7:209-215. Hess, R. A., R. J. Thurston, and H. V. Biellier, 1982. Morphology of the reproductive tract of turkeys producing abnormal yellow semen. Poultry Sci. 61:531-539. Lin, C , and J. Chang, 1975. Electron microscopy of albumin synthesis. Science 190:465—467. Marquez, B. J., and F. X. Ogasawara, 1975. Scanning electron microscope studies of turkey semen. Poultry Sci. 54:1139-1143. O'Rand, M. G., 1974. Isolation and localization of an iron-binding rabbit seminal plasma antigen. J. Reprod. Fert. 39:349-357. Phillips, M., M. W. Olsen, and S. S. Stone, 1973. Bisalbuminemia in the serum of chicken-turkey hybrids. Comp. Biochem. Physiol. 46B:533-539. Saeki, Y., and K. I. Brown, 1962. Effect of abnormal spermatozoa on fertility and hatchability in the turkey. Poultry Sci. 41:1096-1100. Thurston, 1976. Physiopathological studies of semen production in the domestic turkey. Ph.D. dissertation, Univ. Missouri-Columbia. Thurston, R. J., and H. V. Biellier, 1972. Ultrastructure studies of turkey semen. Poultry Sci. 51:1879. Thurston, R. J., R. A. Hess, H. V. Biellier, H. K. Adldinger, and R. F. Solorzano, 1975. Ultrastructure studies of sperm abnormalities and herpes virus associated with cultured testicular cells from domestic turkeys. J. Reprod. Fert. 45:235-241. Thurston, R. J., B. L. Hughes, R. A. Hess, and D. P. Froman, 1982. Seminal plasma free amino acids, and seminal and blood plasma proteins of the guinea fowl (Numidia meleagris). Poultry Sci. 61:1744-1747. Torres-Medina, A., M. B. Rhodes, and H. C. Mussman, 1971. Chicken serum proteins: A comparison of electrophoretic techniques and localization of transferrin. Poultry Sci. 50:1115-1121.
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It was noted that for two YS males whose semen quality approached that of WS (i.e., decreased seminal plasma protein), the percent of total protein represented by albumin (79 and 84% when the protein concentration was 1.3 and 3.1 g/100 ml, respectively) was more than for normal WS (average 61%). Thus, even in supposedly normal WS males, separation of seminal plasma proteins and tests for elevated albumin may permit identification of males with a propensity for YS production. The low correlation between blood and seminal plasma protein concentration unfortunately means that males destined to produce YS cannot be identified by a blood protein concentration test prior to the breeding season.
1911
1982 Poultry Science 61:1905-1911
INTRODUCTION
Yellow turkey semen contains abnormal spermatozoa, spermatids, often numerous macrophages, and has reduced fertilizing capability when compared to normal white semen (Saeki and Brown, 1962; Cherms, 1968; Thurston and Biellier, 1972; Marquez and Ogasawara, 1975; Thurston et ai, 1975). However, the biochemical characteristics of yellow semen are unknown. Preliminary experiments designed to identify the yellow substance showed an elevated concentration of seminal plasma proteins (unpublished data). This finding provided die impetus for the research reported herein. The objectives were 1) to monitor semen quality weekly for 15 weeks (i.e., seminal plasma color, protein concentration and subfractions) and semen volume of males that initially produced white (WS) or
1 Published with approval of die director of the South Carolina Agricultural Experiment Station as Technical Contribution No. 1994.
yellow (YS) semen and 2) to determine the correlation between blood and seminal plasma protein concentration, and 3) to compare the mobility and staining pattern of blood and seminal plasma proteins separated by electrophoresis. MATERIALS AND METHODS
Weekly Evaluation of White Versus Yellow Semen. Large White turkey breeder males (60) were stimulated to produce semen in December with incandescent light (12 lx, 14L:10D). Semen was collected for 5 weeks and evaluated as white or yellow by visual inspection. Eight males producing WS and 8 YS were selected for a further 15-week study of semen quality. All toms were 3 3 weeks old with the exception of two YS males that were approximately 2 years old. The older males were included as Hess (personal communication) found no effect on yellow semen production or characterstics due to age. The males were randomly housed in 1.83 X 2.44 m pens, 4 per pen, and fed a
1905
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ABSTRACT Semen volume, seminal plasma color, protein concentration, and subfractions from 8 white semen (WS) and 8 yellow semen (YS) Large White turkeys were measured for 15 weeks. Average volume was .29 ± .01 ml for WS and .32 ± .02 ml for YS males. Plasma from WS was white and averaged 1.84 ± .07 g/100 ml protein, whereas YS seminal plasma was yellow with 7.03 ± .5 g/100 ml protein. Protein subfractions in WS and YS seminal plasma were qualitatively similar and consisted of prealbumin, albumin, alpha—1 and —2, beta—1, —2, and —3, and postbeta (gamma). The alpha proteins were more frequently seen in YS seminal plasma. The elevated protein of YS seminal plasma was due to an increased concentration of all proteins, with the possible exception of beta—3, with albumin contributing the most. Except for prealbumin and beta—3, the seminal proteins had the same electrophoretic mobility as counterpart blood proteins. Blood and seminal plasma protein concentrations were poorly correlated (+ .039; P<.75). Blood protein concentrations from 68 toms ranged from 3.3 to 5.7 g/100 ml and averaged 4.2 g/100 ml; seminal plasma protein concentrations ranged from .93 to 12.8 g/100 ml. Nineteen percent of the males had YS with seminal plasma protein greater than 4.0 g/100 ml. The high correlation between intensity of the yellow color in YS seminal plasma and protein concentration (+ .76; P<.01) indicates that semen quality of turkey breeders could be improved by selection on the basis of low seminal plasma protein concentration. (Key words.- turkey, blood, semen, plasma, proteins)
1906
THURSTON ET AL.
The protein concentration of frozen-thawed seminal plasma was determined by the biuret method as described by Thurston (1976). A standard curve was prepared using 1 to 16 g/100 ml of chicken blood plasma albumin (Sigma, St. Louis, MO), and a least squares equation of optical density versus albumin concentration was used to determine seminal plasma protein concentration. Only one de-
termination could be made per sample due to the small volume of seminal plasma. Comparison of Seminal and Blood Plasma Proteins. Heparinized (10 units/ml) brachial vein blood and semen were collected from 68 Large White breeder males (42 weeks of age). Plasma was collected by centrifugation and protein concentration was determined by the biuret method, with two measurements per sample. Blood and seminal plasma from 4 WS and 4 YS males were chosen for comparison of plasma protein fractions by electrophoresis; 8 /il of blood plasma were applied per gel. Statistical Analysis. Statistical analysis was with a nested arrangement for analysis of variance, mean differences determined by the method of least significance difference. A model for semen volume by week was determined by the method of least squares. RESULTS
Comparison of Semen Quality of White and Yellow Semen Males. Average semen volume was greater for YS males (P<.05), .32 ± .02 ml versus .29 ± .01 ml for the WS males. The best model for semen volume for the 15 weeks was parallel lines (not rejected at P<.05) with negative slopes (-.006 for YS; - . 0 0 2 for the WS males), indicating that volume slightly declined for both groups as the season progressed. Semen volume of YS toms was greater than for WS males except for 2 weeks. The average seminal plasma color of the YS males was yellow to dark yellow (average code = 3.5 ± .14; Table 1) compared to white (average code = 1.3 ± .10) for WS males. The correlation between degree of yellow color and seminal plasma protein concentration was high + .76 (P«.01), due to increased protein in YS seminal plasma, which averaged 7.03 ± .5 g/100 ml versus 1.84 ± .07 for WS males (P«.05; Table 1). Seminal plasma protein concentration showed little variation by week in the WS males but was more variable for YS males (Fig. 1). This was partially due to grouping on the basis of semen color. The plasma begins to appear yellow when the protein concentration is approximately 2.5 to 3.0 g/100 ml. Thus, males with white semen would have seminal plasma protein concentrations confined to a narrow range (average range was 1.38 to 3.0 g/100 ml), whereas a male classified as YS had an excess of 2.5 to 3.0 g/100 ml, but there was no limit on
Downloaded from http://ps.oxfordjournals.org/ at Albert R. Mann Library on November 1, 2014
turkey breeder ration (16.6% protein; 2641 kcal ME/kg) ad lib. Semen was collected by massage (Burrows and Quinn, 1937) into tuberculin syringes, and volume was recorded. The semen was placed into 5 ml centrifuge tubes and centrifuged at 2000 X g at 4 C for 30 min. Seminal plasma color was estimated (against a light blue background) as white, slight yellow, yellow, or dark yellow and coded 1, 2, 3, or 4, respectively. The plasma was removed and stored in 1 ml vials at 4 C for electrophoresis the following day. Plasma proteins were separated using polyacrylamide disc gel electrophoresis. Gels were polymerized in 11.4 X .5 (i.d.) cm glass tubes, an 8.4 cm, 7% acrylamide separating gel adjusted to pH 8.9 with a Tham-HCl (Fisher Scientific Co., St. Louis, MO) buffer, overlayered with a 2.4 cm, 4% acrylamide stacking gel at pH 6.7 (Tham-HCl buffer). Polymerization of the gel solutions was achieved by adding .01 g/100 ml ammonium persulfate. A Thamglycine buffer, pH 8.3, was used in the cathode and anode chambers. Ten microliters of white or 4 fil of yellow seminal plasma were injected on top of the stacking gel with a microsyringe (except for YS males with <2.5 g/100 ml of protein where 10 /il were added). The remaining plasma was frozen. Electrophoresis was performed using constant voltage; 100 V for 1 hr, then 280 V for 1.5 hr. The gels were stained with amido black for 10 min, electrically destained, cleared with 5% glycerol, and scanned with a Densicord Model 542 densitometer (Photovolt Corp., New York, NY) using visible light. Peak area was integrated as densitometer units by a Photovolt Model 49A integrator. Electrophoresis and densitometer scan of purified turkey seminal plasma albumin showed that peak area (densitometer units) was linearly related to protein concentration between 10 and 230 /Ug of protein (r2 = .96). Therefore, peak area approximated concentration and was used for quantitation of individual protein fractions.
ELEVATED PROTEIN IN YELLOW TURKEY SEMEN
the maximum seminal protein concentration, which was as high as 13.65 g/100 ml. The large variation in seminal plasma protein by week was also due to within male variation. For example, for two males seminal plasma protein declined-, from 4.4 g/100 ml on week 1 to 1.3 g/100 ml at week 15 for one (Fig. 2), and from 10.5 g/100 ml at week 6 to 3.1 g/100 ml at week 15 for the other. Accompanying the diminution in protein was a change in plasma color from yellow to white and slight yellow. None of the WS males changed to chronic YS producers, aldiough two occasionally produced semen with yellow colored plasma and elevated protein. Six of the YS males had elevated seminal plasma protein concentrations for the entire 15 weeks concomitant with yellow or dark yellow plasma, but their protein level varied by week. The most pronounced example of the YS syndrome was exhibited by a male whose protein ranged from 9.4 to 19.5 g/100 ml, and the plasma color was always dark yellow. In contrast, a constant WS male had white plasma whose protein ranged from 1.1 to 1.9 g/100 ml for the 15-week period (Fig. 2). Six major protein fractions were consistently separated from white seminal plasma (Fig. 3). They were identified on the basis of mobility using the nomenclature of Bierer (1969) for turkey serum proteins. In order, beginning with those which migrated farthest from the cathode, were: prealbumin, albumin, beta—1, —2, and —3, and postbeta (gamma) proteins. Alpha—1 and —2 proteins were occasionally detected at low concentrations. Albumin was the most abundant protein, representing 65 to
70% in 6 males, but in the remaining 2 toms with WS it was 50% and 34%. For the latter males, the beta—2 and —3 fractions represented a greater percentage of total protein. The proteins in YS plasma were qualitatively similar to those of WS plasma, but alpha—1, and especially alpha—2, were more frequently observed, particularly in males that had the highest level of seminal plasma protein (Fig. 4). The elevated protein of YS plasma was due to increased concentrations of prealbumin (P<.01), Albumin (P< .01), alpha-2 (P«.05), b e t a - 1 (P<.05), b e t a - 2 (P«.05), and postbeta (P<.05) proteins (Table 1). The average beta—3 peak area for YS (58.4; Table 1) was larger than for WS (45.3; Table 1), but this difference was not statistically significant at (P«.05). Thus all proteins, with the possible exception of beta—3, contributed to the elevated protein in YS seminal plasma. There was a disproportionate contribution to the elevated seminal plasma protein by each of the increased protein fractions in YS (Table 2). The average percent albumin of total seminal protein in six YS males, 81.4%, was 20.3% greater than for WS males, and this is
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FIG. 1. Average seminal plasma protein concentration by week ± SEM. The elevated seminal plasma protein concentration of YS males is evident.
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ELEVATED PROTEIN IN YELLOW TURKEY SEMEN
1909
DISCUSSION Turkey males producing YS have welldeveloped secondary sexual characteristics and
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FIG. 5. Gels depicting blood (top gel) and seminal plasma proteins from a YS male. The scan represents the fractions in blood plasma. 1 = prealbumin; 2 • albumin; 3 = alpha—1; 4 = alpha—2; 5 = beta—1; 6 = beta—2; 7 = b e t a - 3 ; 8 = postbeta (gamma).
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though much more was present than in the seminal plasma of the WS males. This was mainly due to an elevated postbeta fraction, which contributed a greater amount to the total protein than for other YS males. Comparison of Blood with Seminal Plasma Proteins. There was low correlation (+ .039; P<.75) between blood and seminal plasma protein concentrations, which ranged from 3.3 to 5.7 g/100 ml with an average of 4.2 ± .04 g/100 ml for blood and ranged from .93 to 12.8 g/100 ml for seminal plasma. Thirteen of the 68 males (19%) had yellow seminal plasma with protein greater than 4.0 g/100 ml. Eight protein fractions were consistently separated from blood plasma. In order of farthest migration from die cathode they were: prealbumin, albumin, alpha—1 and —2, beta—1, —2, and —3, and postbeta (gamma). For both YS and WS males, the seminal plasma albumin, alpha—1 and —2, and beta—1 and —2 protein fractions generally had the same mobility as corresponding proteins in blood plasma (Fig. 5). Seminal plasma beta—3 migrated differently than the beta—3 protein of blood plasma.
1910
THURSTON ET AL.
The YS was characterized by elevated seminal plasma protein, the concentration highly correlated with the degree of yellow plasma color. It is interesting that Crabo et al. (1971) found that the plasma removed from the epididymis of a bull with abnormal sper-
matozoa also had a higher protein concentration than normal. The yellow color is confined to the plasma (Cherms, 1968), and precipitation of the plasma proteins removes the yellow color, so it is possible that the yellow discoloration is due to the excess protein. The elevated protein was caused by an increased concentration of proteins found 'in normal WS seminal plasma, with the exception of the beta—3. Because most of the elevated YS seminal plasma proteins were similar to blood plasma proteins, blood proteins could possibly get into the seminal fluids and contribute to the increased seminal protein. Alternatively, the protein may be synthesized and released by reproductive tissue. Hess et al. (1982) found that the testes and epididymal region of YS males were yellow. Microscopy revealed that the ductuli efferentes epithelia were hypertrophied and filled with lipid-like vacuoles, and many cells appeared to bleb into the lumen. Lin and Chang (1975) showed that albumin is synthesized by a variety of epithelial types; hence, the excess protein in YS may come from the abnormal efferentes epithelia. Albumin/beta and albumin/postbeta ratios for YS seminal plasma were larger (6 of 8 toms) than for WS, indicating that albumin was disproportionally greater in YS than other proteins. Thus, the elevated protein concentration of YS was not achieved by removal of water, which would concentrate all proteins proportionately. Yellow semen is known to contain abnormal sperm cells and spermatids (Saeki and Brown, 1962; Marquez and Ogasawara, 1975; Thurston et al, 1975), so quantitation of abnormal cells was parenthetical to the objectives of the present research. However, each sample was briefly scored for abnormal cells by microscopic inspection. The number of abnormal cells declined simultaneously with the change in plasma color and decline in seminal plasma protein in the two YS males with transient YS production. These observations, in addition to the fact that the seminal plasma protein of chronic YS males oscillated over time, is evidence that the degree of involvement with the YS syndrome is dynamic. Because of their poor fertilizing capability, current management practices recommend that YS males be culled from breeder flocks. The problem is recognition of the yellow color in whole semen. One can visually detect yellow in the plasma when the protein is approximately 2.5 to 3.0 g/100 ml.
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testes size comparable to those of WS males (Thurston, 1976). These observations and the finding that average semen volume of YS males was slightly more than for WS males for 15 weeks is evidence that the YS syndrome is not the result of refractoriness. The severity of the YS syndrome (degree of seminal plasma protein elevation) was not negatively correlated with semen volume, but the YS male with die most plasma protein (average 13.65 g/100 ml) gave the least volume (average .21 ml) of all YS males. Further research is needed to determine if there is a relationship between semen volume and the degree of plasma protein elevation. The average seminal plasma protein of WS males, 1.84 g/100 ml, was lower than the 2.7 g/100 ml reported by Graham et al. (1971), whose data was obtained from a flock that probably contained YS males. Only 6 major proteins were separated from WS seminal plasma, whereas 14 have been identified in chicken and 9 in guinea seminal plasma (Harris and Sweeney, 1971; Thurston et al, 1982). Based on staining profile and electrophoretic mobility, the turkey seminal plasma proteins, except prealbumin and beta—3, were similar to proteins separated from blood plasma. As for blood, albumin was the most abundant protein, but between male variation in quantity was noted, a possible reflection of different genotypes, as the concentration and phenotype of blood albumin in chickens and chicken-turkey hybrids has been shown to be governed by inherited traits (Fried and Chun, 1971; Phillips et al, 1973). Seminal plasma beta proteins may be transferrin, because their relative mobility and staining pattern were similar to beta—1 and —2 chicken blood plasma transferrins (Glick, 1968; Torres-Medina et al, 1971). B e t a - 3 , the most abundant seminal beta protein, was closely associated with the beta—1 and —2 and may represent transferrin synthesized by cells of the reproductive tract. Similarly, O'Rand (1974) characterized a lactoferrin-like protein in rabbit seminal plasma that differed from blood transferrins. The postbeta fraction was diffuse in WS seminal plasma and probably represents a conglomerate of gamma globulins, possibly antibodies.
ELEVATED PROTEIN IN YELLOW TURKEY SEMEN Plasma protein concentration usually must exceed 4 to 5 g/100 ml before whole semen can be detected as yellow, but acuity is not good in low intensity lighting regimens. Thus, culling of YS males based on visualization of whole semen is subjective with large error. Screening for elevated seminal plasma protein should improve the semen quality of breeder turkey males and help identify semen problems in flocks experiencing idiopathic infertility.
REFERENCES Bierer, B. W., 1969. Serum protein fractions in the normal turkey. Poultry Sci. 48:1208-1215. Burrows, W. H., and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16:19-24. Cherms, F., 1968. Variations in semen quality and the relationship of semen quality to fertility in turkeys. Poultry Sci. 47:746-754. Crabo, B., L. Gustafsson, and A. R. Rao, 1971. Subnormal testicular function in a bull concealed by phagocytosis of abnormal spermatozoa in the efferent ductules. J. Reprod. Fert. 26:393-396. Fried, M., and P. W. Chun, 1971. Polymorphism in fowl serum albumin-I. Characterization of two genetic variants of fowl serum albumin and definition of peptide differences. Comp. Bio-
chem. Physiol. 39B:523-540. Glick, B., 1968. Serum protein electrophoretic patterns in acrylamide gel: patterns from normal and bursaless birds. Poultry Sci. 47:807-814. Graham, E. F., M. L. Schmehl, K. I. Brown, B. G. Crabo, and G. Ertel, 1971. Some chemical studies of the female reproductive tract and seminal plasma of the male turkey and their relationship to fertility. Poultry Sci. 50:1170-1181. Harris, G. C. Jr., and M. J. Sweeney, 1971. Changes in the protein concentration of chicken seminal plasma after rapid freeze-thaw. Cryobiology 7:209-215. Hess, R. A., R. J. Thurston, and H. V. Biellier, 1982. Morphology of the reproductive tract of turkeys producing abnormal yellow semen. Poultry Sci. 61:531-539. Lin, C , and J. Chang, 1975. Electron microscopy of albumin synthesis. Science 190:465—467. Marquez, B. J., and F. X. Ogasawara, 1975. Scanning electron microscope studies of turkey semen. Poultry Sci. 54:1139-1143. O'Rand, M. G., 1974. Isolation and localization of an iron-binding rabbit seminal plasma antigen. J. Reprod. Fert. 39:349-357. Phillips, M., M. W. Olsen, and S. S. Stone, 1973. Bisalbuminemia in the serum of chicken-turkey hybrids. Comp. Biochem. Physiol. 46B:533-539. Saeki, Y., and K. I. Brown, 1962. Effect of abnormal spermatozoa on fertility and hatchability in the turkey. Poultry Sci. 41:1096-1100. Thurston, 1976. Physiopathological studies of semen production in the domestic turkey. Ph.D. dissertation, Univ. Missouri-Columbia. Thurston, R. J., and H. V. Biellier, 1972. Ultrastructure studies of turkey semen. Poultry Sci. 51:1879. Thurston, R. J., R. A. Hess, H. V. Biellier, H. K. Adldinger, and R. F. Solorzano, 1975. Ultrastructure studies of sperm abnormalities and herpes virus associated with cultured testicular cells from domestic turkeys. J. Reprod. Fert. 45:235-241. Thurston, R. J., B. L. Hughes, R. A. Hess, and D. P. Froman, 1982. Seminal plasma free amino acids, and seminal and blood plasma proteins of the guinea fowl (Numidia meleagris). Poultry Sci. 61:1744-1747. Torres-Medina, A., M. B. Rhodes, and H. C. Mussman, 1971. Chicken serum proteins: A comparison of electrophoretic techniques and localization of transferrin. Poultry Sci. 50:1115-1121.
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It was noted that for two YS males whose semen quality approached that of WS (i.e., decreased seminal plasma protein), the percent of total protein represented by albumin (79 and 84% when the protein concentration was 1.3 and 3.1 g/100 ml, respectively) was more than for normal WS (average 61%). Thus, even in supposedly normal WS males, separation of seminal plasma proteins and tests for elevated albumin may permit identification of males with a propensity for YS production. The low correlation between blood and seminal plasma protein concentration unfortunately means that males destined to produce YS cannot be identified by a blood protein concentration test prior to the breeding season.
1911