Published January 20, 2015

Boar sperm quality in lines of pigs selected for either ovulation rate or uterine capacity1,2 B. A. Freking,*3 P. H. Purdy,† S. F. Spiller,† C. S. Welsh,† and H. D. Blackburn† *U.S. Meat Animal Research Center, ARS-USDA, Clay Center, NE 68933; and †National Animal Germplasm Program, Fort Collins, CO 80521.

ABSTRACT: Selection for 11 generations in swine for ovulation rate (OR) or uterine capacity (UC) resulted in significant changes in component traits of litter size. Our objective was to conserve the unique germplasm for the future and to characterize sperm quality as a correlated response to the selection criterion imposed compared with an unselected control line (CO). Boars representing genetic diversity available in all 3 lines were produced in 2 farrowing seasons. Season 1 was born in September 2005 and was sampled for semen characteristics in October 2006. Season 2 was born in March 2006 and was sampled for semen characteristics in February and March 2007. Each boar (n = 60) was collected twice. The sperm-rich fraction was obtained, and volume and concentration of sperm cells were measured to estimate total sperm production. Each ejaculate was extended 1:3 (vol/vol) with Androhep Plus (Minitube, Verona, WI) and was packed for shipping to the National Animal Germplasm Program laboratory for processing into frozen straws. Semen quality was measured by computer-assisted semen analysis at 3 semen processing points: fresh (FR), 24 h after extender added (E), and postthaw (PT). A mixed model ANOVA was applied to the

data. Fixed effects of farrowing season, line, and 2-way interactions were fitted. The random effect of boar (n = 60) within farrowing season and line was used to test line differences. Sperm concentration was not different (P = 0.18) among the lines (0.594 × 109, 0.691 × 109, and 0.676 × 109 cells/mL for CO, OR, and UC lines, respectively). However, significance (P = 0.04) was detected for the volume of the sperm-rich fraction, greatest for OR (86.4 mL), intermediate for UC (75.5 mL), and least for CO (70.2 mL). Line differences were thus detected (P = 0.02) for total sperm production per ejaculate, greatest for OR (54.9 × 109), intermediate for UC (48.7 × 109), and least for CO (40.5 × 109). A larger percentage of progressively motile sperm and greater estimates of sperm velocity only at processing point E (P < 0.01) were detected in favor of CO. Estimates of motility, velocity, and other parameters of sperm movement measured on E processing points were positively correlated with the same estimates obtained PT, but the magnitude was low to moderate (r range −0.03 to 0.23). Thus, selection for component traits of female reproduction had a favorable effect on total sperm production of boars.

Key words: fertility, ovulation rate, pig, semen quality, uterine capacity © 2012 American Society of Animal Science. All rights reserved.

J. Anim. Sci. 2012.90:2515–2523 doi:10.2527/jas2012-4723 INTRODUCTION

1Mention of trade names is necessary to report factually on avail-

able data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the same by USDA implies no approval of the product to the exclusion of others that may also be suitable. The USDA is an equal opportunity provider and employer. 2The authors acknowledge, at the U.S. Meat Animal Research Center, the expert technical assistance of A. Kruger for semen evaluations, the swine crew for expert animal husbandry led by R. Pooschke and D. Porter, and J. Watts for secretarial support. 3Corresponding author: [email protected] Received September 19, 2011. Accepted February 7, 2012.

Boar fertility has a major impact on reproductive efficiency of swine operations. Male fertility can be influenced by the number and quality of spermatozoa capable of fertilization. The primary determinant of daily sperm production in boars is the number of Sertoli cells, which establishes testicular weight (Ford et al., 2006). There is considerable biological variation in sperm production between and within breeds and lines of pigs (Kennedy and Wilkins, 1984; Okwun et

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al., 1996; Flowers, 2008), which could be exploited to improve fertility. Single-trait selection for 11 generations in swine for ovulation rate (OR) or uterine capacity (UC) resulted in significant changes in component traits of litter size (Leymaster and Christenson, 2000; Freking et al., 2007). It is unknown how this selection affected male fertility. Boar fertility responded favorably to selection for testes size by increasing sperm production per ejaculate and decreasing age when boars reached their plateau for maximum daily sperm production (Rathje et al., 1995; Huang and Johnson, 1996). Early efforts (Land, 1973) suggested strong genetic correlations between reproductive traits such as testes weight in males to OR in females. Single-trait selection for OR resulted in increased testes weight by 10% to 12% at 140 to 160 d of age (Schinckel et al., 1983). In contrast, selection for testes weight did not increase OR in pigs (Johnson et al., 1994). These observations provide convincing evidence for a strong genetic component to boar fertility, yet the correlated effect on female reproduction traits has been inconsistent. This paper reports sperm quality measurement recorded from lines of pigs selected for component traits of female reproduction: OR and UC. Our 2-fold objective was to estimate line differences in sperm quality as a correlated response and to provide samples to facilitate long-term storage of these unique germplasm resources. MATERIALS AND METHODS All experimental procedures were approved and performed in accordance with U.S. Meat Animal Research Center (USMARC) Animal Care Guidelines and the Guide for Care and Use of Agricultural Animals in Agricultural Research and Teaching [Federation of Animal Science Societies (FASS), 1999]. Population Description A 4-breed composite with equal contributions from Chester White, Landrace, Large White, and Yorkshire breeds was formed to estimate breed, heterosis, and recombination effects on growth, carcass, and reproductive traits (Cassady et al., 2002a,b). From a common base generation of this composite produced in 1986, selection was initiated during 1988 within 2 replicated seasons for increased OR estimated from laparoscopic examination of gilts in the OR line, increased litter size of unilateralhysterectomy-ovariectomy surgically altered gilts (defined as UC) in the UC line and a randomly selected control line (CO). The selection experiment proceeded for 11 generations until 1999 when all 3 lines were evaluated for responses in selected component traits and litter size (Leymaster and Christenson, 2000). Relative to

CO, selection for OR increased OR by 3.2 ova, decreased UC by 0.97 pigs, decreased prenatal survival by 10.3%, and increased litter size by 0.30 pigs. Selection for UC increased OR by 0.13 ova, increased UC by 2.15 pigs, increased prenatal survival by 3.5%, and increased litter size by 0.62 pigs. In a separate evaluation initiated after relaxed selection for 5 generations, it was shown that intact UC at day 114 of gestation estimated from survival curves predicted values of 14.0, 11.8, and 15.6 live fetuses for CO, OR, and UC lines, respectively (Freking et al., 2007). Selection for UC improved fetal survival relative to the other lines primarily during the time period between days 25 and 45 of gestation (Freking et al., 2007). Magnitude of line differences in UC were essentially established by day 45, and all 3 lines exhibited fetal losses at a similar rate throughout the remainder of gestation. After the 11-generation selection experiment, each line was maintained, with no intentional selection pressure, in 2 genetic replicate farrowing seasons that annually occurred in either March or September. Consequently, genetic replicate and season were completely confounded. Genetic diversity within each selection line and season was maintained by an intended family size of 10 boars and 40 gilts farrowed for each line. Boars representing the 10 sire lines within each population were sampled from 2 replicate farrowing seasons. Sample 1 was born in September 2005 and was evaluated for semen characteristics in October 2006. Sample 2 was born in March 2006 and was evaluated for semen characteristics in February and March 2007. Semen Collection Each boar had experienced a previous natural mating breeding season and produced progeny. A collection before the experiment was discarded during the training process, but boars were collected using females in estrus rather than a collection dummy. Each boar (n = 60) was collected at least twice using the gloved-hand technique (n = 137 observations; Table 1), with approximately 7 to 10 d between collections. Boars were selected at random with respect to lines for the date of first collection, ensuring balanced numbers across lines for a given date. Boars were penned in individual stalls (approximately 50 cm × 213 cm) until time of collection when they were transported to Table 1. Number of boars and ejaculates sampled for semen characteristics by line and season

Selection line Control Ovulation rate Uterine capacity

Sampling season October February to March No. of No. of No. of No. of boars ejaculates boars ejaculates 10 20 10 28 11 21 10 25 9 18 10 25

Sperm quality selected for maternal traits

a pen (244 cm × 244 cm) in a separate building containing a female in standing estrus. Because the primary objective was cryopreservation, only the sperm-rich fraction of the ejaculate was obtained, and volume and concentration were measured to estimate total sperm production. At this time, a sample was obtained for analysis of motility and quality of the fresh ejaculate. The remaining ejaculate was then extended 1:3 (vol/vol) with Androhep Plus (Minitube), warmed to 37°C and prepared for shipping to the National Animal Germplasm Program (NAGP) laboratory for further processing into frozen straws. The sample was poured into 50-mL conical tubes and placed in a container with room-temperature water (23°C). The sample was allowed to cool to 23°C over 1.5 to 2.0 h by placing the container with the sample and room-temperature water on the laboratory bench out of direct light. Once the sample reached room temperature, the sample was transferred to a shipping cooler set to 15°C. After reaching 15°C, the sample was placed in a prepared shipping box and transported overnight to the NAGP laboratory. Semen Processing at NAGP Samples were processed essentially as outlined in Purdy (2008). Upon arrival at NAGP, samples were centrifuged at 800 × g for 10 min at 15°C and the supernatant was removed. The sperm in the resulting pellet, before cryopreservation, will be referred to as the afterextended sample. Sperm concentration was determined using a spectrophotometer (Spectronic 20 Genesys, Spectronic Instruments, Rochester, NY) pecifically calibrated for boar sperm (Foote, 1972). Boar sperm samples were diluted using BF5 cooling extender [CE; 52 mM TES, 16.5 mM tris (hydroxymethyl) aminomethane, 178 mM glucose, 20% egg yolk; 325 mOsm] in two-thirds of the final volume and cooled to 5°C over 2.5 h (Pursel and Johnson, 1975). Samples were then diluted with BF5 freezing extender [91.5% CE, 6% glycerol, 2.5% Equex Paste (Minitube), vol/vol; one-third of the final volume; 1450 mOsm (Pursel and Johnson, 1975)] so that the final sperm concentration was 200 × 106 sperm/mL and loaded into 0.5-mL CBS straws (IMV Corporation, Minneapolis, MN). The samples were frozen in liquid nitrogen vapor using a Minidigitcool UJ400 programmable freezer (IMV Corporation) with the following freeze rates: 5°C to −8°C at −20°C/min, −8°C to −120°C at −69°C/min, −120°C to −140°C at −20°C/ min. The straws were then plunged into liquid nitrogen for storage. Samples were thawed, usually the next day, by submerging a semen straw in a 50°C water bath and gently agitating the straw for 20 s. Thereafter, the straws were maintained at 37°C and processed immediately for postthaw analysis.

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Description of Computer-Assisted Semen Analysis Traits Quantitative measures of sperm production analyzed included volume of collection (mL), concentration (109 sperm/mL), and total sperm production per ejaculate (volume × concentration). Using computer-assisted sperm analysis (CASA) critical velocity, average path velocity, straight-line velocity, amplitude of linear head displacement, beat cross frequency, straightness, and linearity were measured as an assessment of semen quality, according to previous reports (Farrell et al., 1998; Hirano et al., 2001). Semen quality was evaluated independently at 3 processing points: fresh (FR), 24 h after extender added (E), and postthaw (PT). Evaluation at time FR was done at USMARC using SpermVision software (Minitube). Evaluations at time E and PT were conducted at NAGP using a Hamilton Thorne Motility Analyzer (Beverly, MA). We recognize that no attempt was undertaken to standardize values between the 2 machines. Measures of sperm quality include evaluations of both sperm cell motility and cell morphology. The following traits were exported from the CASA evaluation of each boar sample. Sperm velocity traveled was estimated using 3 path descriptions: according to the actual curved-line path (VCL in μm/s), the straight-line path (VSL in μm/s), and the average smoothed path (VAP in μm/s). Unitless ratios of velocity measures were used to define sperm path movement in terms of path curvature as follows: linearity (LIN), calculated as VSL/VCL, and straightness (STR), calculated as VSL/VAP. Amplitude of lateral head displacement (ALH) was used to estimate the distance of side-to-side movement of sperm head. Beat cross frequency (BCF) was the frequency of the flagella beat in hertz. Elongation (ELONG) was a unitless ratio of sperm head width:length used as a measure of sperm morphology. Motility (MOT) was defined as the percentage of total sperm cells where VCL exceeded the threshold rate of 10 μm/s. Progressive motility (PMOT) was used to classify movement as a fraction of all sperm moving in a progressive path defined as VCL > 25 μm/s and STR > 80%. The CASA was set up as follows: 30 frames acquired, frame rate of 60 Hz, minimum contrast of 55, minimum cell size of 5 pixels, VAP cutoff of 20 μm/s, progressive minimum VAP cutoff of 45 μm/s, VSL cutoff of 5 μm/s, static head size of 0.53 to 4.45, and magnification of 1.89 (which were preset by the manufacturer). A minimum of 7 fields and 1,000 sperm were observed for motility analysis. Statistical Analyses Data were analyzed with the mixed-model ANOVA procedure (SAS Inst. Inc., Cary, NC). Each trait defined above was considered a trait of the individual boar, and for semen quality, each measurement and processing point combination was analyzed as a unique trait. Fixed

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effects of season (1 and 2), line (CO, OR, and UC), and 2-way interactions were fitted. The random effect of boar (n = 60) within season and line was used to test the line mean square. The Kenward-Roger option was used to approximate denominator degrees of freedom associated with the random effect of boar. Preliminary models tested the linear covariate of number of days between ejaculate collections, but this was not significant (P > 0.05) for any trait analyzed and was subsequently dropped from the final models. When line was a significant (P < 0.05) source of variation, linear contrasts were constructed to separate line means. Partial correlations were obtained using the MANOVA/printe option in SAS GLM after accounting for line and season effects in the model.

similarly to the means for volume, line differences were detected (P = 0.02) for the product of the 2 traits: Total sperm production per ejaculate. Reflecting the observed line differences in volume, total sperm production per ejaculate was greatest for OR (54.9 × 109), intermediate for UC (48.7 × 109), and least for CO (40.5 × 109). CASA Measures of Sperm Motility and Velocity

Levels of significance and least-squares means are reported for effects of selection line (Tables 2 to 4). Estimated differences between lines are the result of combined direct additive, dominance, and any epistatic genetic effects that represent the full line differences for the response variables. All 3 populations would be expected to express similar levels of heterosis at the beginning of the selection experiment. Variance component estimates presented consist of both between-boar variance and within-boar variance from the multiple collections obtained. Large variance components for the between-boar estimate relative to the within-boar estimate would indicate the importance of individual boar differences. If the within-boar variance estimate is relatively large, it would indicate a larger amount of variation within each boar for the multiple samples taken.

Results for analysis of sperm motility and velocity measures are tabulated by selection line and processing point of measurement (Table 3). Measures of sperm velocity represented by VSL, VCL, and VAP displayed differences between lines (P < 0.05) only for the E processing point. In all 3 of these instances CO line estimates of sperm velocity were greater than either of the other 2 lines, which were similar to each other. Estimates obtained at FR and PT processing points did not differ among the 3 lines. Among the unitless ratios of velocity measures used to define sperm path movement, line differences were detected (P < 0.01) only for STR, calculated as VSL/VAP, and only for the E processing point. In this instance, the CO population ratio of VSL relative to VAP was less (less straight) than observed in OR and UC lines. The ratio LIN approached significance (P = 0.06) for line, but differences among means were small. Differences among lines were detected (P < 0.05) for MOT and PMOT at the E processing point only. Mean MOT was greater for the CO (85.1% exceeded VCL of 10 μm/s) line than either of the selected lines. As expected, most measures of motility and velocity collected at the PT processing point tended to be less than measures estimated from FR and E processing points.

Sperm Production

Measures of Sperm Activity and Morphology

Results for analysis of sperm production traits are presented in Table 2. Selection line differences were detected (P = 0.04) for volume of the sperm-rich fraction, with means greatest for OR (86.4 mL), intermediate for UC (75.6 mL), and least for CO (70.2 mL). Sperm concentration was not statistically different (P = 0.17) among the lines (0.594 × 109, 0.691 × 109, and 0.676 × 109 cells/mL for CO, OR, and UC lines, respectively). However, because means for concentration were ranked

Results of analysis of sperm motility and morphology traits are tabulated by selection line (Table 4). Differences were detected (P = 0.04) among lines for BCF at the FR processing point only. Less activity was observed for the UC line (36.5 Hz) relative to the CO (40.0 Hz) and OR (39.6 Hz) lines. The main caveat to this observation is that this measure is a frequency of the actual track path crossing the smoothed average track, and when the sperm path is highly circular, these

RESULTS General

Table 2. Boar selection line least-squares means, SE, and levels of significance for total sperm production traits per ejaculate Selection line Control Ovulation rate Trait Volume, mL 70.2 ± 4.5a 86.4 ± 4.5b Concentration, × 109/mL 0.594 ± 0.039 0.691 ± 0.038 Total sperm production, × 109 40.5 ± 3.5a 54.9 ± 3.4b a,bWithin a row, means without a common superscript differ (P < 0.05).

Uterine capacity 75.6 ± 4.8a,b 0.676 ± 0.040 48.7 ± 3.6a,b

P-value for line 0.04 0.18 0.02

Variance component estimates Between boar Within boar 123.7 655.2 0.015 0.033 124.5 260.1

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Table 3. Boar selection line least-squares means, SE, and levels of significance for sperm motility, velocity, and velocity ratio measures on fresh semen after extended and postthaw processing points Trait Motility, % Fresh Extended Postthaw Progressive motility, % Fresh Extended Postthaw Velocity straight line (VSL), μm/s Fresh Extended Postthaw Velocity curved line (VCL), μm/s Fresh Extended Postthaw Velocity average path (VAP), μm/s Fresh Extended

Control

Selection line Ovulation rate

Uterine capacity

P-value for line

Variance component estimates Between boar Within boar

88.3 ± 2.7 85.1 ± 1.7a 39.6 ± 3.1

87.3 ± 2.7 76.2 ± 1.7b 30.5 ± 3.0

80.3 ± 2.8 74.4 ± 1.9b 34.2 ± 3.3

0.10 0.0001 0.11

122.5 19.3 105.4

55.9 70.4 147.8

81.1 ± 3.1 44.4 ± 1.9a 18.7 ± 2.0

79.8 ± 3.0 41.7 ± 1.9a,b 13.6 ± 1.9

71.3 ± 3.2 37.2 ± 2.1b 17.7 ± 2.1

0.07 0.05 0.16

155.3 31.3 37.3

75.5 78.4 72.9

57.5 ± 1.9 55.5 ± 1.5a 55.0 ± 1.4

57.3 ± 1.9 52.6 ± 1.5a,b 55.4 ± 1.4

51.7 ± 2.0 50.0 ± 1.6b 56.7 ± 1.6

0.07 0.05 0.72

42.2 19.9 18.8

62.3 45.6 42.2

300.3 243.4 154.9

255.9 464.6 259.4

125.7 ± 4.5 220.0 ± 5.0a 144.5 ± 3.9

129.8 ± 4.4 191.6 ± 4.9 b 141.5 ± 3.8

71.3 ± 2.3 106.0 ± 2.7a

72.4 ± 2.3

66.0 ± 2.4

0.14

71.7

81.3

91.0 ± 2.7b 73.0 ± 1.9

88.8 ± 3.0b 74.3 ± 2.1

0.0001 0.82

67.7 39.5

147.8 60.4

0.43 ± 0.009 0.27 ± 0.006 0.40 ± 0.008

0.18 0.06 0.39

0.0009 0.0004 0.0003

0.0009 0.0003 0.0016

0.78 ± 0.007 0.57 ± 0.011b 0.74 ± 0.010

0.11 0.002 0.30

0.0005 0.0012 0.0006

0.0011 0.0012 0.0019

Postthaw 74.6 ± 1.9 Linearity, VSL/VCL Fresh 0.45 ± 0.008 0.44 ± 0.008 Extended 0.26 ± 0.006 0.28 ± 0.005 Postthaw 0.39 ± 0.008 0.40 ± 0.007 Straightness, VSL/VAP Fresh 0.80 ± 0.007 0.79 ± 0.007 Extended 0.53 ± 0.010a 0.58 ± 0.010b Postthaw 0.72 ± 0.009 0.73 ± 0.009 a,b Within a row, means without a common superscript differ (P < 0.05).

values are not considered valid as an intended measure of activity. The CO line was observed to have greater (P = 0.03) ALH, which measures side-to-side movement of the sperm head, than either the OR line or the UC line at the E processing point only. Data collected at processing points FR and PT did not detect line differences. The ratio ELONG was not measured at the FR processing point and was not significantly different (P ≥ 0.08) among lines at the other 2 processing points. This unitless ratio of sperm head width:length indicated this measure of overall sperm morphology was similar across lines. Correlations among Traits and across Times Because total sperm production would be of primary importance to boar studs, the correlations of total sperm production per ejaculate with estimates of motility, activity, and morphology are presented in Table 5. As one would expect, the component traits of volume and concentration were highly correlated to total sperm produc-

119.8 ± 4.7 190.9 ± 5.5b 142.7 ± 4.2

0.30 0.0001 0.86

tion. Measures of motility and velocity describing different path movements were not correlated with total sperm production. Correlations between the 2 ratio measures describing sperm movement paths and total sperm production were inconsistent in direction and significance among the different processing points. The ratio LIN was not correlated with total sperm production at the E processing point but was negatively correlated with total sperm production at the FR and PT processing points. The ratio STR was also not correlated with total sperm production at the E processing point but was negatively correlated at FR and PT processing points. For measures of sperm activity, BCF was negatively correlated with total sperm production at E and PT processing points. In contrast, ALH movement was positively correlated with total sperm production at FR and PT processing points. Table 6 presents correlation coefficients to address whether observations taken at the normal processing point after the addition of semen extenders are indicative of PT motility and activity. For traits MOT, PMOT, VSL, VCL, VAP, LIN, STR, and BCF there were no observed

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Table 4. Selection line least-squares means, SE, and levels of significance for sperm activity and morphology measures on fresh semen after extended and post-thaw processing points Selection line Trait Control Ovulation rate Beat cross frequency, Hz Fresh 40.0 ± 1.0a 39.6 ± 1.0a Extended 30.6 ± 0.5 31.3 ± 0.5 Postthaw 36.4 ± 0.5 37.1 ± 0.5 Amplitude lateral head displacement, μm Fresh 3.00 ± 0.09 3.17 ± 0.09 Extended 8.92 ± 0.12a 8.49 ± 0.12b Postthaw 6.76 ± 0.17 6.55 ± 0.17 Elongation (head width:head length), % Fresh — — Extended 54.3 ± 0.55 53.2 ± 0.54 Postthaw 49.9 ± 0.41 48.9 ± 0.40 a,b Within a row, means without a common superscript differ (P < 0.05).

significant correlations between the E and PT processing points (r range of −0.03 to 0.18). A similar correlation for ALH was significant (P = 0.02) and positive but relatively moderate in strength (r = 0.23). In general, correlation values among the different traits, motility, and activity measurements taken within a processing point exhibited moderate to large correlations with each other, whereas correlations across the processing points were much smaller in magnitude. Storage of Frozen Semen Individual boar and population description information is publicly available for this conserved unique germplasm at http://www.ars.usda.gov/Main/ Table 5. Partial phenotypic correlations (r) of total sperm production with estimates of motility and sperm activity at the 3 processing points

Trait Volume, mL Concentration, × 109/mL Motility, % Progressive motility, % Velocity straight line (VSL), μm/s Velocity curved line (VCL), μm/s Velocity average path (VAP), μm/s Linearity, VSL/VCL Straightness, VSL/VAP Beat cross frequency, Hz Amplitude lateral head displacement, μm * P ≤ 0.05 for H0 r = 0. ** P ≤ 0.01 for H0 r = 0. *** P ≤ 0.001 for H0 r = 0.

r with total sperm production, × 109 Fresh Extended Postthaw 0.54*** 0.52*** −0.01 0.13 −0.03 −0.06 0.14 −0.01 0.01 0.11 −0.08 0.12 0.05 0.05 0.10 0.08 0.01 −0.19* 0.09 −0.23* −0.31*** 0.03 −0.28** −0.17* −0.18 −0.26** 0.27*** 0.09 0.30**

Uterine capacity

Level of significance for line

Variance component estimates Between boar Within boar

36.5 ± 1.0b 31.9 ± 0.6 36.4 ± 0.5

0.04 0.25 0.51

3.09 ± 0.09 8.54 ± 0.13b 6.69 ± 0.19

0.40 0.03 0.68

0.103 0.128 0.254

0.135 0.282 0.643

— 52.5 ± 0.60 49.0 ± 0.45

— 0.08 0.18

— 4.18 0.04

— 3.21 6.41

17.3 2.3 1.4

5.6 5.5 6.8

docs.htm?docid=16979. A total of 22,970 straws (0.5 mL) of semen were processed and contributed to the NAGP inventory of composite populations of pigs. These lines currently represent >10% of all swine semen in the NAGP inventory and almost 30% of composite semen samples. DISCUSSION Improvement in boar fertility can result from increasing numbers of sperm produced or improving their ability to fertilize eggs. As the swine industry has evolved into greater use of AI, a better understanding of the genetic basis for the tremendous amount of variation among boars and between lines of boars would be of economic value to swine producers and boar studs. Industry selection among and within lines for reproduction has logically focused more on female reproductive traits. Although the industry has done a remarkable job in evaluating breeds or lines and appropriately establishing roles for these lines in crossbreeding systems to optimize performance (Cassady et al., 2004; Moeller et al., 2004; Serenius et al., 2006), it is not entirely clear what the correlated impact on male reproduction has been. If the effect is negative in response to improved female reproductive selection, this would generate a negative economic impact on the components of the industry producing replacement females. Data from selection experiments in mice and sheep (Land, 1973) indicated increased OR associated with larger testes and provided the motivation for a selection experiment in pigs for testes weight (Johnson et al., 1994) as a mechanism to indirectly achieve greater OR in females. However, after 10 generations of selection, these researchers observed correlated responses in OR and age at puberty that were small and nonsignificant. Islam et al. (1976) reported that OR of mice were

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Table 6. Partial phenotypic correlations among extended and postthaw processing points for semen motility and activity Point1 Column 1 2 3 4 5 6 7 8 9 E 1 PT 2 0.142 Progressive motility E 3 0.78 0.19 PT 4 0.13 0.95 0.18 Velocity straight line E 5 0.59 0.22 0.73 0.19 PT 6 −0.09 0.36 0.02 0.50 −0.01 Velocity curved line E 7 0.60 0.14 0.34 0.12 0.68 −0.07 PT 8 0.03 0.40 0.08 0.51 0.14 0.73 0.14 Velocity average path E 9 0.63 0.15 0.39 0.13 0.77 −0.06 0.97 0.15 PT 10 0.02 0.45 0.08 0.57 0.11 0.90 0.09 0.94 0.11 Linearity E 11 0.13 0.15 0.57 0.14 0.55 0.12 −0.21 0.08 −0.05 PT 12 −0.20 −0.02 −0.13 0.02 −0.26 0.38 −0.28 −0.32 −0.29 Straightness E 13 −0.13 0.15 0.43 0.14 0.25 0.13 −0.47 0.02 −0.40 PT 14 −0.25 −0.07 −0.18 −0.03 −0.32 0.33 −0.33 −0.29 −0.36 Beat cross frequency E 15 −0.22 0.14 −0.15 0.11 −0.08 0.08 0.04 0.02 −0.08 PT 16 −0.24 −0.11 −0.27 −0.13 −0.32 0.08 −0.20 −0.37 −0.24 ALH3 E 17 0.35 0.12 0.06 0.08 0.53 −0.09 0.79 0.13 0.78 PT 0.18 0.16 0.19 0.20 0.28 0.14 0.23 0.63 0.27 1E = processing point after extender added; PT = postthaw processing point after cryopreservation. 2 P ≤ 0.05 for H r = 0when │r│ > 0.20. 0 3 ALH = amplitude lateral head displacement. Trait Motility

increased by selection for testes weight. Additionally, Eisen and Johnson (1981) observed strong genetic correlations in male reproductive traits represented by testes weight in mice selected for litter size of females. Unique breed differences also contribute to the inconsistent correlations between male and female fertility observed in pigs. Meishan boars reach puberty at an earlier age and have smaller testes compared with commercial crossbred boars available to U.S. swine producers, yet OR and litter size of Meishan females are large (Borg et al., 1993; Christenson et al., 1993; Lunstra et al., 1997). Comparing different breeds, Young et al. (1986) found that the genetic relationships of testicular traits with age at puberty and litter size of females were not consistent in magnitude or sign. In the current experiment, a line representing an increased OR of 3 to 4 ova resulted in a small but favorable response in total sperm production, whereas selection for UC without changes in OR resulted in intermediate but greater levels relative to CO. Thus, direct selection of females for independent component traits of litter size as represented by OR and UC lines resulted in small but positive changes in male fertility in terms of total sperm production. No information has been collected on testes weight in these lines. Total sperm production per ejaculate in the pig populations used in this experiment was not correlated with measures of sperm motility and movement. Information generated by CASA evaluation has been employed to predict human semen quality and, when combined with subjective relative quality score information, predicts

10

11

12

13

0.09 −0.03 0.04 −0.08 0.04 −0.22 0.07 0.47

−0.03 0.85 −0.06 −0.12 −0.22 −0.23 0.14

0.11 0.92 0.08 0.69 −0.30 −0.77

0.12 0.09 −0.08 −0.47 −0.01

14

15

16

17

0.10 0.74 0.18 −0.33 −0.17 −0.26 −0.79 −0.11 −0.82 0.23

reproductive success (Agarwal et al., 2003). A multitrait index using information obtained through CASA was developed and associated with pregnancy success under assisted reproductive technologies (Bedaiwy et al., 2003). Semen evaluation by CASA is used by the swine industry, but it remains unclear what variables are useful to improve selection for boar fertility (Holt et al., 1997; Didion, 2008). On the basis of a large survey reported by Knox et al. (2008), semen of boar studs is typically evaluated for motility within 0 to 5 min of warming in an extender with viewing at 100× to 400× magnification. Sperm concentration estimation typically occurs by using a spectrophotometer, and CASA and ejaculate collections are discarded for reasons of poor motility, abnormal sperm, and bacterial contamination. The utility of various semen characteristics for selection to improve fertility warrants further investigation. Genetic variation in sperm cell quality among and within industry standard composite populations was reported (Stewart et al., 2006). These authors concluded that variation in quality traits and motion parameters among 4 genetic lines at 2 studs was not significant among lines and among collections, but the effects of stud and individual boar variation were large. Similar to what was observed in the current study, significant decreases in motility and most measures of individual sperm activity decreased in PT semen relative to E semen. On the basis of CASA values reported by Schmidt and Kamp (2004), this may be indicative of hyperactivation, but certainly indicates an altered PT sperm qual-

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ity. These authors also observed that progressive motility estimated from FR semen was a significant predictor of PT progressive motility. Smital et al. (2004) showed that progressive motility in pigs was moderately heritable and correlated with the litter size of females mated. In the current experiment, motility differences detected among lines in the extended semen were not manifested as line differences in PT estimates, and the correlations observed between E and PT processing points were small to moderate in magnitude. It seems clear that these estimates of motility from PT processing points would cause challenges in any attempt to regenerate these lines of pigs using only frozen semen and traditional backcrossing, although low motility could be compensated for by using more straws. Although line differences were detected in the current experiment for some sperm activity measures using CASA-derived traits, the differences were not large or consistent across processing points. Differences between individual boars were much larger than what was observed for average line differences. Motility and velocity traits for the selected populations in this study were less than for the CO line, suggesting lowered sperm movement when estimated at the E time point, but this did not translate into line differences when estimated at the PT time point after the cryopreservation protocols. This also suggests that not all lines of pigs, or even individual boars within lines, react similarly to the same extender or holding times for cryopreservation. Information obtained in this study would indicate that the selection criterion for increased OR or UC imposed on females has resulted in modest increased sperm production in males, with minor changes in sperm quality or movement. These data suggest further work in developing accurate predictors of PT sperm performance and its ability to produce viable pregnancies is needed. LITERATURE CITED Agarwal, A., R. K. Sharma, and D. R. Nelson. 2003. New semen quality scores developed by principal component analysis of semen characteristics. J. Androl. 24:343–352. Bedaiwy, M. A., R. K. Sharma, T. K. Alhussaini, M. S. Mohamed, A. M. Abdel-Aleem, D. R. Nelson, A. J. Thomas Jr., and A. Agarwal. 2003. The use of novel semen quality scores to predict pregnancy in couples with male-factor infertility undergoing intrauterine insemination. J. Androl. 24:353–360. Borg, K. E., D. D. Lunstra, and R. K. Christenson. 1993. Semen characteristics, testicular size, and reproductive hormone concentrations in mature Duroc, Meishan, Fengjing, and Minzhu boars. Biol. Reprod. 49:515–521. Cassady, J. P., O. W. Robison, R. K. Johnson, J. W. Mabry, L. L. Christian, M. D. Tokach, R. K. Miller, and R. N. Goodwin. 2004. National Pork Producers Council maternal line genetic evaluation: A comparison of growth and carcass traits in terminal progeny. J. Anim. Sci. 82:3482–3485.

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