COMPARISON OF THE SEMEN CHARACI’ERISTICS OF FENGIING, MEISHAN AND YORKSHIRE BOARS R.W. Gerfen,l
B.R. White,1 M.A. Cotta2 and M.B. Wheeler1.a 1Laboratory of Molecular Embryology Department of Animal Sciences University of Illinois Urbana, lL 61801, USA 2Fermentation Biochemistry Agricultural Research Service, USDA Peoria, IL 61604, USA
Received for publication: ApriZ 27, 1993 Accepted: October 19, 1993 ABSTRACT Semen characteristics of Chinese Fengjing, Chinese Meishan and American Yorkshite boars were examined. Samples were collected from 24 boars: 6 Fengjing, 12 Meishan and 6 Yorkshire. Three semen chara&ristics and 6 biochemical evaluations of semen or seminal plasma were analyzed. The whole semen parameters measured were gelatinous (gel) and gel-free volume and progressive motility. Fengjing boars were higher in gel volume than Meishan (P < 0.05) but not Yorkshire boars (P > 0.25), while Yorkshire boars were higher than Meishan boars in gel volume (P < 0.10). The gel-free volume was higher in Yorkshire and Fengjing boars than Meishan boars (P c O.lO), but Fending gel-free volumes did not differ from Yorkshire gel-free volumes (P > 0.80). However, the only difference detected for progressive motility was between Fengiing and Meishan boars (78.5 vs 74.5%; P < 0.10). Sperm concentration was higher in Meishan than Yorkshire boars (P c 0.01) although these breeds did not differ from Fengiing boars (P > 0.18). The biochemical measurements made were fructose, galactose, inositol, total carbohydrate and total protein in seminal plasma and pH in gel-free semen. Fengjing boars had higher concentrations of galactose in seminal plasma than Meishan or Yorkshire boars (P < 0.05). while Meishan seminrd plasma had higher galactose concentrations than York&ire seminal plasma (P < 0.10). Fmctose, inositol and total carbohydrate concentrations were all higher in Fengjing and Me&an seminal plasma than Yorkshire seminal plasma (P < 0.05). Fengiing seminal plasma had higher levels of inositol than Meishan seminal plasma (769 vs 566 mg/lOO ml, P < 0.10). Furthermom, Fengjing and Meishan semen had similar fructose and total carbohydrate concentrations (P > 0.80). The pH level and total protein concentration did not differ among the 3 breeds (P > 0.30). This study indicated that breed differences were detected for several seminal parameters, although no single breed had consistantly higher values for all the parameters measured. Key words: semen, boar, Chinese pigs, seminal plasma Acknowledgements This study was supported, in part, by grants from the University of Illinois Agricultural Experiment Station (IL 35-0380) and the Illinois Pork Producers Association (90-PRI-IJvI1000). The authors gratefully acknowledge Ling-Ling Lo, Diana Bidner, Gary Edmonds and Bill Fisher for their technical expertise and assistance. a&respondence and reprint requests.
Copyright 0 1994 Butterworth-Heinemann
Certain breeds of Chinese swine have been recognized for their increased prolitlcacy (19,41). The Chinese Meishan and Fengjing breeds have been shown to have larger litters than the occidental Yorkshire breed (25,37). To manipulate the reproduction of these breeds successfully, it is essential to understand their reproductive physiology, including whether or not Chinese boar breeds have reproductive traits similar to those of occidental breeds. In swine, the size of the litter is dependent upon the ovulation rate, fertilization rate, embryonic mortality and uterine capacity (38). The boar affects the fertilization rate through the quality and quantity of semen deposited in the female (39). The most commonly used laboratory test for evaluating semen is visual estimation of the percentage of motile spetmatozoa (10). Although motile spermatozoa are requited for normal fertility, motility alone is not a guarantee of fertility. The quantity of spermatozoa also plays a role in determining the fertilization rate in the pig (34). In addition, the biochemical composition of boar semen influences fertility through effects on sperm viability and female reproductive tract environment (9). The objective of our study was to determine whether Chinese Fengjing, Chinese Meishan and American Yorkshire breeds differ in semen characteristics and composition. MATERIALS AND MRTHODS Animals and Semen Collection Sexually mature Fengjing (n=6) and Meishan (n=l2) boars, ranging in age from 6 to 34 mo, and Yorkshire (n=6) boars, at 10 mo of age, were used for semen collection. All the boars were housed in a environmentally controlled building in individual sow gestation crates under artificial lighting. The boars were fed a balanced ration, and water was available ad libitum (14). Semen was collected from the boars using the gloved hand technique (16), either on a sow in standing estrus or on a phmtom mount. All the boars were allowed at least 3 days of sexual rest between collections (21) and between 1 and 8 collections per boar were analyzed. All fractions of the semen were collected into clean Styrofoam cups, and only complete collections were analyzed. Sperm and Seminal Plasma Analyses Whole semen was transported to the laboratory in an insulated thermos. The gel fraction was separated from the semen by filtration through cheese cloth (16). The gel and gel-free semen volumes were measured in a lOOO-ml graduated cylinder. A 15ml aliquot of gel-free semen was immediately frozen at -20 ‘C for fructose, galactose, inositol, total carbohydrate and pH analyses. Progressive motility of the spermatozoa was assessed visually - 10 mitt after semen collection under a light microscope.a Sperm concentration was determined with a specuophotometer according to methods described by Gebauer et al. (12). Semen pH was measured with a pH meter using a combination e1ectrode.b Seminal plasma was separated from the spermatozoa by cenuifugation 3 times at 600 x g for 20 min. The seminal plasma was frozen at -20 ‘C until analyzed. A bicinchoninic acid protein assayc was used for the spectrophotometrid determination of total seminal plasma protein concentration in microtiter plates (22,32).
aNikon Corp., Tokyo, Japan. bBeckman Instruments, Inc., Brea, CA. cPierce Chemical Co., Rockford, IL. dBIO-mR Instruments, Inc., Winooski, VT.
Theriogenology Analyses of Seminal Plasma Carbohydrate, Fructose, Galactose and Inositol
Seminal plasma samples were subjected to ultrafiltration through Millipore Ultrafree-MC filter unitse at 2000 x g and filtrates analyzed for total carbohydrate, fructose, galactose and inositol content. The total carbohydrate level of filtered samples was determined using the anthronesulfuric acid assay (11). Fructose content was determined using a modification of the sucrose assay procedure described by Russell and Baldwin (27). Filtered seminal plasma (0.045 ml) and 0.9 ml of an enzyme-buffer solution (0.5 M triethanolamine, pH 7.5, containing per ml, hexokinaselglucose-6phosphate dehydmgenase, 2.5 units; phosphoglucose isomerase, 5 units; MgClz6H20.0.7 mg; and ATP, 0.35 mg), and 0.045 ml NADP solution (10 mg/ml in H20). The reduction of NADP was monitored spectrophotomettically at 340 nm for 30 min, and the increase in absorbance was compared to that for a fructose standard (0 to 750 [email protected]
).f The galactose and inositol contents of the seminal plasma were determined by gas-liquid chromatography of alditol acetates prepared by the procedure of Albersheim et al. (1) as modified by Stack (33). Samples were. reduced with sodium bomhydtide and acetylated with acetic anhydride and dimethylamino pyridine. The resultant alditol acetates were separated on a DB-225 fused silica capillary columng in a gas chromatographh heated to 210 ‘C. Peaks detected by flame ionization were processed by an integrator’ and identified by comparison with retention times of authentic standards. The identity of neutral sugars was further confirmed by thin layer chromatography of seminal plasma filtrates on Whatman silica gel plates (K-5; 250-pm layer thickness), a solvent of 91 acetonitrile and water, and spots visualized by reaction with N-(lnaphthyl)ethylenediamine dihydrochloride at 100 “C (5). statistical Analyses Semen parameters were statistically evaluated using the following general linear model (29) for calculating trait means of gel volume, gel-free semen pH, and seminal plasma fructose, galactose and total protein. Statistical differences were determined using the t-test for pairwise significant differences: Yijk
Bi + G(B)ij +
where yi& = an observable random (dependent) variable; p = an unknown constant; Bi = fixed effect of the i* breed (Fengiing, Meishan, Yorkshire); G(B)ij = fixed effect of the jth boar (j=1,2,3,...,12) nested within the ie breed, l31 = partial linear regression of the dependent variable on age of boar, xi%; B2 = partial linear regression of the dependent variable on collection date, zis; eijk
iid N (O,& e).
The semen parameter for fructose and total carbohydrate content were logarithmically transformed to reduce inequality of variances between breeds (30). Gel-free volume, progressive motility, sperm concentration, total carbohydrate and inositol data were statistically evaluated using the following general linear model (29):
e30,OOOMW cutoff, Millipore Corp., Bedford, MA. fAll materials from Sigma Chemical Co., St. Louis, MO. gJ&W Scientific, Ranch0 Cordova, CA. h589OA, Hewlett-Packard, San Fernando, CA. i3392A, Hewlett-Packard, Palo Alto, CA.
Bi + G(B)ij +
+ &rZi& + ejl,
where yi# = an observable random (dependent) variable; CL= an unknown constant; Bi = fixed effect of the ih breed (Fengjing, Meishan, Yorkshire); G(B)ij = fixed effect of the jh boar (j=1,2,3,...,12) nested within the ia breed; I31= partial linear regression of the dependent variable on age of boar, xi&; St I= quadratic regression of the dependent variable on age of boar, Xi&; & = partial linear regression of the dependent variable on collection date, z+ q% = random residual, e+#‘s - iid N (O,a* e). RESULTS Shown below (Table 1) are trait variations among the 3 breeds of boars. Differences in age of the boar at collection among the breeds was primarily due to the 8-mo quarantine period of the Chinese boars (19). To prevent disease and to maintain similar housing conditions for all the study animals, Yorkshire boars were raised in the swine facility reserved for the Chinese boars and hence they were younger at collection than the Chinese boars. Heterogeneity of variance among the 3 breeds in fructose and total carbohydrate concentrations probably amse from differences in age and the number of samples analyzed. Table 1. Summary of breed means for semen and seminal plasma traits in Chinese Meishan, Chinese Fengjing and American Yorkshire boars
Fenaiina Mean SD
6 14 15 16 15 16 16 16 18 14 17
762 133 95.4 45.6 143.0 36.9 75.6 2.4 989.6 305.2 24.9 17.4 39.0 16.1 535.4 227.4 7.94 0.33 58.4 42.8 28.4 11.8
Gel volume (ml) Gel-free volume (ml) Progressive motility (%) Sperm concentration (1OVml) Fructose (mg/lOO ml) Galactose (mg/loO ml) InositoI (mg/lOO ml) pH (gel-tree semen) Total carbohydrate (mg/lOO ml) Total protein (mg/ml)
186 13.4 35.3
;; 24 74.0 26 1001.0 37.0 ;; 30.5 27 501.3 7.94 ;: 79.1 31 34.5
546j65 43.; 13.3 228.2 0.24 49.8 12.3
orks e n yMeat!r SD 287 29 95.0 44.1 238.0 110.2 76.0 4.9 657.1 235.7 10.4 8.3 24.8 11.4 501.3 262.4 7.77 0.14 28.7 18.2 29.4 11.2
Whole Semen Characteristics The volume of gelatinous material in Fengjing boar semen was higher than that in the Meishan boars (P < 0.05; Table 2) but not in Yorkshire boar semen (P > 0.25). Further, the gel volume of Yorkshire boars was higher than than that of Meishan boars (P c 0.10). The semen of Yorkshire and Fengiing boars had a higher gel-free volume than the semen of Meishan boars (P < 0.10); however, the gel-free volumes of Fengjing boar semen did not differ from that of the Yorkshire
In addition, the percentage of ptogressively motile spermatoxoa was higher for (P > 0.80). Meishan than for Fen&g boars (P < 0.10). although Yorkshire boars did not differ from Meishan or Fengjing boars in progressive motility (P > 0.27). The concentration of spermataxoa was higher for Meishan than for Yorkshire boars (P c 0.01). but Fending boars did not differ from the other 2 breeds for sperm concentration (P > 0.18). boars
Table 2. Least squares means f SEM for semen traits of Chinese Meishan, Chinese Fengjing and American Yorkshire boars Trait
Gel volume (ml) Gel-free volume (ml) Progressive motility (%) Sperm concentration (106/ml)
111.1 176.3 78.6 957.6
f f f f
19.6ac 28.3c 2.3c i24.5a.b
47.3 132.8 74.5 1061.4
f f + f
7.8bd 13.3d 1.4d 70.2a
75.9 f 186.6 f 74.3 + 671.7 +
12.7abc 19.4c 1.8cd lo4.3b
a,b Means in the same row with different superscripts differ (P < 0.05). c,d Means in the same row with different superscripts differ (P c 0.10). Table 3. Least squares means f SEM for the biochemical composition of Semen and seminal plasma from Chinese Meishan, Chinese Fengjing and American Yorkshire boars Trait
Fructose (ms/lOO ml)a Galactosc (mg/lOO ml) Inositol (mg/lOO ml) pH (gel-free semen) Total carbl_ohydrate (mg/lOO ml)a Total protein (mg/ml)
28.9 f o.lb 44.0 f 5.9bd 7;;; r ;1_3bd 99.0 f 155.7b 27.2 f 7.2
25.7 + o.lb 30.3 + 3.0cc 565.6 f 68.Obc 7.81 f 0.04 107.8 it 124.8b 34.8 f 3.1
5.1 io.1c 18.6 + 4.8Cf 253.2 It 103.7cf 7.82 f 0.07 9.8 f 143.5c 30.6 f 5.6
a Geometric means. b,c Means in the same row with different superscripts differ (P < 0.05). d.e,fMeans in the same row with different superscripts differ (P < 0.10). Biochemical Analyses of Seminal Plasma Data for fructose and total carbohydrate concentrations were logarithmically transformed to reduce the variation that existed among breeds for these traits (Table 1). The values that resulted from the transformation are within the reported ranges. Fructose, inositol and total carbohydrate concentrations were all higher in Fengjing and Meishan seminal plasma than in Yorkshire seminal plasma (P < 0.05; Table 3). Fengjing and Meishan biochemical parameters were not different (P > 0.30) except for inositol(769 vs 566 mg/lOO ml, P < 0.10) and galactose (44 vs 30 mg/loO ml, P < 0.05) concentrations, which were higher in the Fengjing seminal plasma. The concentration of
galactoseinMeishan seminal plasma was greater than that in Yorkshire seminal plasma (P < 0.10). Finally, gel-free semen pH and the concentration of total ptotein in the seminal plasma were not significantly different among the 3 breeds (P > 0.30). DISCUSSION The prolifIcacy of the Chinese Fengjing and Meishan swine breeds has lead to several studies examining the role of the female in attainment of large litter sixes (2,4). However, few studies have investigated the role that the Chinese boar may play in this regard. Several studies on occidental swine bteeds have shown breed differences in seminal characteristics (15.35). The results from those studies indicated that no single breed is superior in all reproductive parameters. In addition, breed comparisons for boar seminal traits can differ from study to study as a result of differences in sampling of boars and in experimental methods (15). The Fengjing and Meishan boars were significantly different with respect to gel volumes (P < 0.05), and Yorkshim gel volumes were higher than Meishan gel voIumes (P < 0.10). Gel from the Chinese breeds of boars was similar to that of Yorkshire boars in appearance, mostly in the form of mu&oval be&s. The observed gel volumes were comparable to the gel volumes mported by Cameron (677) for occidental breeds. The gel-free volumes were higher in Yorkshire and Fengjing boars than in Meishan boars (P < O.lO), but the gel-free volumes of Fen&g boars did not differ from those of Yorkshire boars. Although, since the Yorkshire boars were only 9 mo of age, gelfree volumes from the Yorkshire boars might have been larger had the Yorkshire boars been 12 mo of age. Semen volume may have important implications in the reproductive function of swine since large volumes Q 50 ml) are required for mechanical stimulation of uterine contractions (34). The values we report for all 3 breeds were well above this minimum, and thus no major difference in mechanical stimulation of the uterus was expected between the breeds. In 1990, a study by Christenson and Lunstm (8) comparing Fengjing, Meishan, Minzhu and Duroc boars showed differences (P < 0.01) between the 4 breeds for gel-free semen volumes, with Duroc boars having the largest volume. The volume of gel-free semen in our study was similar to that reported by Swierstra (35) for Yorkshire boars and slightly higher than that reported by Christenson and Lunstra (8) for Fen&g and Meishan boars. ‘Ihe largest single ejaculate of gel-f& semen in our study for a Fengjing boar was 210 ml; it was 175 ml for a Meishan and 550 ml for a Yorkshire boar. Chinese boar spermatozoa had a similar percentage of motility as Yorkshire spermatozoa, However, Fengjing and Meishan spermatazoa did differ in progressive motility. This difference was small (78.6 vs 74.5%; P < 0. lo), and the subjective natum of this measurement may have influenced the nsults. The progressive motility scotes we obtained are similar to those teported by Swierstra and Rahnefeld (36). These results ate also supported by the study of Christenson and Lunsua (8) which did not find significant differences in progressive motility scores among Fengjing, Meishan and Dutoc boars. Further, spermatozoa from Chinese boars appeamd [email protected]
similar to Yorkshire spermatozoa, which supports the results of Christenson and Lunstra (8). who found no morphological differences between Fengjing, Meishan and Dumc spermatozoa We determined that Meishan boars had higher concentrations of spennatazoa than York&ire boars (P c 0.01); however sperm concentrations from Fengjing boars did not differ i?om those of Meishan and Yorkshire boars. ‘Ihe biochemical composition of seminal plasma may be infkrenced by many factors. All the samples used in our study were collected under similar conditions and by one person in order to minimiz the effects ofcollection and handling. Seminal plasma of the boar is known to be rich in inositol(3.0 to 4.5 mg/ml) but it has a much lower content of fructose (9 to 60 mg/lOO ml) than that of either the bull or the ram (3). In addition, boar seminal plasma also contains an appreciable quantity of galactose (4 to 20 mg/lOO ml), which in combination with inositol and f?uctose cunp&es most of the free carbohydrates in the seminal plasma (3). The level of fructose observed
in our study was similar to that obtained by Baronos (3) in occidental seminal plasma Fructose is principally secreted from the seminal vesicles in the boar and thus is an indicator of the seminal vesicle contribution to whole semen (18). Based upon this parameter, Fending and Meishan semen may have larger seminal vesicle contribution to whole semen than Yorkshire semen since both have significantly higher (-25.7 vs 5.1 mg/lOO ml) fructose concentrations. However, this can only be confirmed thmugh split fraction semen collection and analysis. Finally, the values we repart for inositol, galactose and total carbohydrate are similar to those obtained by Baronos (3), with small differences likely due to the method of analysis. The significantly higher csoncentrations of fructose, inositol and total carbohydrate in Fending and Meishan seminal plasma may influence overall fertility. However, since motility is morn highly correlated with fertility, is generally a better indicator of fertility and was not different between the Chinese breeds of boars and the Yorkshire boars, the importance of carbohydrate and sugar is unclear. The pH of semen plays an important role in the initiation of spexm motility (23). The pH values we sport 8n slightly higher (7.82 vs 7.61) than those reported by Rodriguez-Martinez et al. (24). This difference may be the consequence of the methods of measuring pH (electrode vs acid/base analysis) and of differences in the handling of semen. Finally, the concentration of protein in the seminal plasma may have an effect on spermatozoa. In the boar, the seminal vesicles secrete most of the proteins in the seminal plasma (17). Most of the proteins secreted by the seminal vesicles are basic in nature (28) and may play a role in maintainiig the sperm plasma membrane. The concentrations of total proteins in the seminal plasma reported in our study were similar among the 3 breeds and corresponded to those reported by Setchell and Brooks (31) for domestic breeds and by Ruixang (26) for the Fengjing breed. For all semen measurements, except for motility and pH, we observed a large within- and between-boar variation. Such variation has been previously described for boars (15) with repeatability factors of semen traits across breeds ranging from 0.21 to 0.30. Thus, the variations found in this study may be intrinsic to boars and not necessarily all based on the experimental methods used. Although this study indicated breed differences in several semen parametets, other parameters were similar among the breeds and indicated no major differences among the Fengiing, Meishan and Yorkshire boars. In conclusion, the mechanism of attainment of large litters is largely unknown in the Fending and Meishan Chinese breeds. The results of our present study suggest that several semen parameters differ between Chinese and occidental (Yorkshire) breeds. The concentrations of fructose and galactose in Chinese boar semen may be beneficial to understanding fertility differences in boars. The concentration of inositol, which is quite high in boar semen, may also provide some clues to boar fertility, since inositol may play a role in redating the osmotic pn~sure of boar semen (13). Finally, the amount of protein in the seminal fluid may be important for stabiliig the plasma membrane of spermatozoa (20) and thus may affect the ability of spermatozoa to fertilize ova. However, further investigation is required to determine the precise effect of the biochemical differences in the seminal plasma on fertility. REFERENCES 1. AlbersheimP, Nevins DJ, English PD. Karr A. A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohyd Res 1967; 5:340-345. 2. Ashworth CJ, Haley CS, Aitken RP, Wilmut I. Embryo survival and conceptus growth after reciprocal ;;~.ryo transfer between Chinese Meishan and Landrace x Large White gilts. 1 Reprod Fertil 1990; 90:5953. Baronos S. Seminal carbohydrate in boar and stallion. J Reprod Fertil 1971; 24303-305. 4. Bazer FW, Thatcher WW, Martinat-Botte F, Terqui M. Sexual maturation and morphological development in Large White tid prolific Chinese Meishan pigs. J Reprod Fertil 1988; 83:723-728. 5. Bounias M. N-(l-naphthyl)ethylenediamine dihychloride as a new reagent for nanomole quantification of
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