Introduction

With regard to evaluation of male fertility, the swine industry is faced with two problems. First, at the present time, there are no accurate tests for boar fertility that can be completed within a reasonable time frame. Available microscopic and biochemical tests for semen quality are primarily indicators of sperm viability and poor estimates of fertilizational competence. These tests can be used to identify extremely infertile males, but are not sensitive enough to discriminate between fertility of boars within normal ranges of motility (> 60%) and morphology (> 70%). Individual boar performance records can be used to determine fertility of boars, but to be useful, require large numbers (> 100) of homospermic matings from sows on the same farm that are bred by a single technician - a situation that doesn’t occur often in practical production situations. Second, there is significant variation in fertility among boars that appear to have a normal complement of spermatozoa.

In other species, protein markers are present in seminal plasma that exhibit either a strong positive or negative relationship with male fertility. The relationship between these proteins and fertility has been examined most extensively in dairy bulls. In dairy bulls, there are proteins that are prevalent in semen from bulls of above average fertility and different proteins that are abundant in seminal fluids from bulls of below average fertility. Boars also exhibit unique, individual differences in the profile of their seminal plasma proteins. Whether or not these proteins can be used as an estimate of the fertility of boar semen is not known and the rationale for pursuing the present study.

Objective

To determine the relationship between seminal plasma protein profiles and semen fertility in boars under controlled experimental conditions.

Materials and Methods

Ten mature boars were collected once per week for 26 weeks. Collections began in the middle of August and were completed by the end of January. Seminal plasma protein profiles of each ejaculate were determined using two-dimensional polyacrylamide gel electrophoresis with isoelectrical focusing and densotometry. In addition, each ejaculate was evaluated for commonly used semen parameters such as motility, mophology and acrosin activity.

Semen from each ejaculate was processed for in vitro fertilization procedures and used to fertilize mature porcine eggs according to established procedures. For each ejaculate from each boar, five different doses of spermatozoa - 10, 10³, 10⁵, 10⁷ and 10⁹ - were used to fertilize eggs (n=50/dose/boar; n=250/boar). Examination of the fertilization rate over a wide range of sperm numbers is necessary to accurately evaluate fertility in vitro because previous studies indicate that determination of in vitro fertilization rates in this manner are positively correlated to farrowing rates and litter size in pigs.

A second series of experiments also was conducted in order to provide in vivo estimates of boar fertility. The best fertility test for boars is a situation in which equal numbers of spermatozoa from different boars are deposited in a female simultaneously and allowed to compete during fertilization. The most fertile boar should fertilize the most eggs and, thus, sire the most pigs in a litter. Such a situation is possible with heterospermic situations and DNA fingerprinting techniques. Based on the relative amounts of the seminal plasma proteins boars were ranked from 1 to 10. Comparisons between boars were conducted in such a way that boars with high concentrations of the a specific protein were compared to those with low concentrations of the same protein. This was done by mixing equal numbers of spermatozoa from two boars and inseminating sows. Blood samples were collected from each boar, sow and piglet that was born alive. Analyses of restriction endonuclease fingerprints (DNA fingerprints) was used to determine the sire of each pig in each litter.

Results and Discussion

Data concerning relationships between the four seminal plasma proteins and in vitro fertilization rates are shown in Table 1. For simplicity, the 10 boar were numbered 1 through 10 based on their arithmatic ranking of in vitro fertilization. Based on this ranking scheme, the 10 boars fell into four distinct fertility classes: > 90% - boars 1 and 2; 80 to 89% - boars 3 and 4; 60 to 79% - boars 5, 6, 7; and < 60% - boars 8, 9 and 10. The relative amounts of the 26 kDa, pI 6.2 protein are nearly perfectly correlated with in vitro fertilization rates in that the boars in the > 90% fertility group have the highest relative concentrations of this protein while boars in the lower fertility groups have progressively smaller amounts of this protein. The same general trend is true for the 55 kDa, pI 4.5 protein. These data indicate that the two proteins - 26 kDa, pI 6.2; and 55 kDa, pI 4.5 - exhibit a positive relationship with in vitro estimates of fertility and have potential for use as a proactive indicator of semen fertility. Furthermore, there were no significant differences among boars in terms of the percentage of motile and morphologically normal spermatozoa or acrosin activity. The mean for all boars during the experiment were 83. 5 + 5.2 %, 88.9 + 6.2% and 92.3 + 8.7% for motility, normal morphology and normal acrosin activity, respectively.

^a,b,c,d,emeans within the same column with different superscripts differ (p < .05)

There was not a significant effect of season on the concentrations of any of the fertility proteins measured. This statement is based upon the lack of an effect of time or week during the study. However, it is important to realize that boars were collected only once per week and this began in the middle of August. It is possible that under a more intense collection regimen seasonal effects may be observed.

Results from four pairwise in vivo comparisons between boars whose ejaculates contain different amounts of the two proteins correlated with in vitro fertility are shown in Table 2. It is important to remember that, in theory, when equal numbers of spermatozoa are mixed from two boars and inseminated into sows at the same time, then the male with more fertile spermatozoa would be expected to fertilize a larger percentage of eggs and, thus, sire more piglets in the litter compared to his less fertile counterpart.

^aboar numbers based on in vitro fertilization rates and concentrations of 26 kDa protein.
^btotal numbers of pigs born alive from farrowings of 5 to 6 sows

These data concerning the relationship between concentrations of seminal plasma proteins and in vivo estimates of fertility are exciting. In the first three comparisons, the boar with the lower number contained the highest concentration of proteins and sired about 90% of the pigs. In the fourth comparison, (boar 1 vs boar 2), in vitro fertilization and concentrations of fertility proteins were not different between the two boars and the percentage of the litter sired by each boar was about 50%. Thus, it appears that under ideal experimental conditions, concentrations of seminal plasma proteins, also, appear to be highly correlated with in vivo fertility.

Summary

Boars with high concentrations of at least two seminal plasma proteins had higher in vitro and in vivo fertilization rates compared to boars with low concentrations of these proteins even though semen characteristics such as motility and morphology were not different. Measurement of these proteins in seminal plasma have the potential to serve as the basis of a proactive fertility test.