Swine News March, 2000 · Volume 23, Number 2

NCSU Extension Swine Husbandry1999
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March, 2000 ^. Volume 23, Number 2

February Swine News

IMPROVING FERTILITY WITH SEMINAL PLASMA

Introduction

At the end of 1999, nearly 60 percent of all the sows in the United States had been artificially inseminated. One of the primary benefits of AI is that a single collection of semen can service a multitude of females. Normally, a single boar ejaculate that is collected on the farm consists of a large volume of fluid (seminal plasma) and a number of spermatozoa (50-100 billion). Even though a single ejaculate contains high numbers of sperm, only 2 to 5 billion are required in an AI dose of semen to achieve successful fertility rates. After sperm are counted in a semen collection, the number of AI doses that can be produced is calculated by dividing the number of sperm by the desired number per dose (i.e. 50 billion by 2.5 billion per dose = 20 doses). Liquid semen extender is then added to the ejaculate to produce 20 doses consisting of at least 60 milliliter which optimizes sperm transport. This process of semen extension is standard throughout the AI industry; however, I will present evidence that will suggest that in addition to (low sperm numbers), overextension of seminal plasma during this process reduces the potential fertility of each semen dose. I will also suggest semen processing methods to capture the fertility enhancement that seminal plasma appears to have.

Background

I have focused my research during the past five years on developing strategies to enhance the viability of sperm inside the reproductive tract with the hopes of improving the efficiency of multiple insemination use in swine AI. From this research we have discovered that the fluid portion of a boar ejaculate (seminal plasma), which is extensively removed through dilution during the semen extension process, has potential fertility benefits that are often overlooked. The number of AI doses that can be produced from a boar ejaculate is currently calculated from the estimated number of motile sperm in that ejaculate. Most producers dilute boar ejaculates with semen extender so that the final AI dose consists of 2 to 5 billion motile sperm cells. However, our research suggests that when diluting semen, the volume of semen plasma should also be considered.

Seminal plasma appears to be involved in ovulation advancement, improved sperm transport, and indirect fertility enhancements. In addition to these potential benefits, our investigations suggest that seminal plasma is also important for sustaining sperm viability during uterine transport and may negate the potential effects a poor uterine environment may have on spermatozoa and developing embryos. Sperm are only viable in the female tract for 12 to 36 hours. Because of this short lifespan, multiple inseminations are commonly performed so that at least one of these inseminations occurs at an optimal (maximum fertilization) time relative to ovulation. Sows and gilts can have estrus periods that last up to 4 days, and although ovulation generally occurs approximately 40 hours after the onset of estrus, both of these parameters are difficult to estimate ahead of time. Therefore, methods to extend the fertile lifespans of sperm in the female would perhaps allow for fewer inseminations since AI could occur at the beginning of estrus regardless of when the female ovulates after the insemination. It seems that one of the major factors limiting the viability of sperm in the female tract and reducing in inseminations per estrus, is a naturally occurring inflammatory response to semen after every breeding. This response seems to be the primary mechanism to clean and prepare the uterus for embryo development and attachment.

Our laboratory studies have indicated that 1) a minimal amount of seminal plasma is necessary in a dose of semen to sustain the fertility of the spermatozoa after deposition into the female, and 2) seminal plasma in an AI dose is important in controlling uterine inflammation. When semen is deposited directly into the uterus, it causes a rapid influx of white blood cells (PMNs) into the uterine lumen. In the presence of seminal plasma, PMNs are rapidly cleared from the uterus. In the absence of seminal plasma via extensive dilution, more PMNs migrate into the lumen and persist for as long as 36 h. This appears to be of great importance when performing multiple inseminations, because if an insemination is performed inadvertently during the end of estrus, a reduction in fertility may occur due to a persisting inflammatory response caused by that AI. We have also recently reported results from a study that examined the effect that repeated or multiple inseminations with or without seminal plasma can have on each other. It seems that the fertility of a second or third AI can be impaired if deposited during a persistent inflammatory response caused by the preceding insemination. This is important to consider since in most AI protocols, the timing of the second service is closest to ovulation. Preliminary data from our laboratory suggests that at least 10 to 12 percent of the entire AI dose should consist of seminal plasma to protect and improve the viability of spermatozoa once in the female reproductive tract. Therefore, we suggest that boar producers collect the entire ejaculate because the fluid portion of an ejaculate is seminal plasma. I offer two methods of semen processing to ensure adequate volumes of seminal plasma in an AI dose of semen (an 85-milliliter dose consisting of 3 billion sperm cells).

Adjusting the Final Dose Volume to Ensure Adequate Amounts of Seminal Plasma

When a boar produces an ejaculate volume of 200 milliliter that has 100 billion motile cells, roughly 33 doses of semen will be made from that ejaculate (100 ÷ 3 = 33 doses). Normally, 2605 milliliter of semen extender is added to the ejaculate and divided equally into 33 containers (33 doses x 85 ml - 200 ml = 2605). In this scenario, each dose contains about 6 milliliter of raw semen or approximately 7 percent seminal plasma. I suggest that instead of adding 2605 milliliter of semen extender, add only 1780 milliliter of extender so that the final AI dose consists of 60 milliliter of fluid, containing 10 percent raw semen or approximately 10 percent seminal plasma. On the other hand, some boars produce larger volumes of semen and thus, final AI does will be larger in total volume. For example, AI semen doses from a boar that gives a 450-milliliter ejaculate containing 80 billion motile sperm cells will each contain 17 milliliter of neat semen (80 ÷ 3 = 26; 450 ÷ 26 = 17). Instead of making 75-milliliter AI doses, add 2150-milliliter of semen extender to this ejaculate, resulting in 100-milliliter AI doses that contain 17 percent seminal plasma. When diluting the semen, consider the volume of ejaculated semen that goes into the final dose. The tables illustrate this dilution scheme from boars with different ejaculate volumes and concentrations of semen for producing semen doses containing 2.5, 3.5, and 4.0 billion spermatozoa.

Table Instructions

Collect semen and measure volume and total number of sperm.
Estimate motility (i.e. 85 percent) and multiply this factor by the total number of sperm. This is the total number of motile sperm in the collection.
Determine the number of sperm you wish to inseminate with (i.e., 2.5, 3.5, or 4.0 billion) and use the respective table to determine the dilution rate.
On the left column of the table find the collection volume that most closely matches the measured volume of the ejaculate.
Scroll to the right until you reach the column representing the total number of motile sperm cells that you have calculated from step two.
Dilute the ejaculate with the semen extender amount indicated (shown in bold type) and the suggested dose volume (under the extender value) into the desired number of doses (column heading shown in parentheses).
Follow suggestions under table for collections that are either too dilute (TD) or too concentrated (TC) for producing AI doses with at least 10 percent seminal plasma.

Table 1. The amount of extender (bold type) added (final dose volume in parentheses) to dilute boar collections for producing AI doses of semen containing 2.5 billion spermatozoa with at least 10 percent of the volume consisting of seminal plasma.^{a, b}
	Total Motile Sperm Cells in Ejaculate (1 x 10⁹) (Number of Potential AI Doses)
Ejaculate Volume	40 (16)	50 (20)	60 (24)	70 (28)	80 (32)	90 (36)	100 (40)	110 (44)	120 (48)
100	940 (65)	TC^NP	TC^NP	TC^NP	TC^NP	TC^NP	TC^NP	TC^NP	TC^NP
150	1210 (85)	1180 (65)	1290 (60)	TC^NP	TC^NP	TC^NP	TC^NP	TC^NP	TC^NP
200	1160 (85)	1500 (85)	1840 (85)	1760 (70)	1720 (60)	TC^NP	TC^NP	TC^NP	TC^NP
250	1350 (100)	1450 (85)	1790 (85)	1880 (85)	2300 (80)	2270 (70)	2150 (60)	TC^NP	TC^NP
300	1300 (100)	1700 (100)	1740 (85)	1830 (85)	2420 (85)	2460 (85)	2700 (75)	2560 (65)	TC^NP
350	1250 (100)	1650 (100)	2050 (100)	1780 (85)	2370 (85)	2410 (85)	3050 (85)	3170 (80)	3010 (70)
400	TD^NP	1600 (100)	2000 (100)	2400 (100)	2320 (85)	2360 (85)	3000 (85)	3340 (85)	3440 (80)
450	TD^NP	TD^NP	1950 (100)	2450 (100)	2750 (100)	2310 (85)	2950 (85)	3290 (85)	3390 (85)
500	TD^NP	TD^NP	TD^NP	2400 (100)	2650 (100)	3100 (100)	2900 (85)	3240 (85)	3340 (85)
550	TD^NP	TD^NP	TD^NP	2350 (100)	2600 (100)	3000 (100)	2850 (85)	3190 (85)	3290 (85)
^a Volumes are in milliliter. ^b 85-milliliter is considered an optimal dose volume whereas volumes below 60 milliliter are sub-optimal for sperm transport. TC^NP indicates that the collection is too concentrated to dilute for making AI doses at 2.5 billion per dose. Final AI doses of more than 60 milliliter with 10 percent seminal plasma are not possible. Previously collected seminal plasma can be added to these doses (5-10 milliliter per dose) to compensate for the lack of seminal fluid in the collection. TD^NP Indicates that the collection is too dilute and although final AI doses will contain more than 10 percent seminal plasma, the final dilution rate will be less than 1 part semen to 5 parts extender. It is not recommended to store this semen for an extended time. Semen may be used immediately or for seminal plasma collection.

Table 2. The amount of extender (bold type) added (final dose volume in parentheses) to dilute boar collections for producing AI doses of semen containing 3.5 billion spermatozoa with at least 10 percent of the volume consisting of seminal plasma.^{a, b}
	Total Motile Sperm Cells in Ejaculate (1 x 10⁹) (Number of Potential AI Doses)
Ejaculate Volume	40 (12)	50 (14)	60 (17)	70 (20)	80 (22)	90 (26)	100 (29)	110 (31)	120 (34)
100	920 (85)	880 (70)	TC^NP	TC^NP	TC^NP	TC^NP	TC^NP	TC^NP	TC^NP
150	870 (85)	1040 (85)	1295 (85)	1350 (75)	1280 (65)	TC^NP	TC^NP	TC^NP	TC^NP
200	1000 (100)	1200 (100)	1245 (85)	1500 (85)	1670 (85)	1750 (75)	1830 (70)	TC^NP	TC^NP
250	TD^NP	1150 (100)	1500 (100)	1450 (85)	1620 (85)	1960 (85)	2215 (85)	2230 (80)	TC^NP
300	TD^NP	TD^NP	1450 (100)	1700 (100)	1580 (85)	1910 (85)	2165 (85)	2180 (80)	2080 (70)
350	TD^NP	TD^NP	TD^NP	1650 (100)	1850 (100)	2250 (100)	2115 (85)	2285 (85)	2540 (85)
400	TD^NP	TD^NP	TD^NP	TD^NP	1800 (100)	2200 (100)	2500 (100)	2235 (85)	2490 (85)
450	TD^NP	TD^NP	TD^NP	TD^NP	1750 (100)	2150 (100)	2450 (100)	2650 (100)	2440 (85)
500	TD^NP	TD^NP	TD^NP	TD^NP	TD^NP	2100 (100)	2400 (100)	2600 (100)	2900 (100)
550	TD^NP	TD^NP	TD^NP	TD^NP	TD^NP	TD^NP	2350 (100)	2550 (100)	2850 (100)
^a Volumes are in milliliter. ^b 85-milliliter is considered an optimal dose volume whereas volumes below 60 milliliter are sub-optimal for sperm transport. TC^NP indicates that the collection is too concentrated to dilute for making AI doses at 3.5 billion per dose. Final AI doses of more than 60 milliliter with 10 percent seminal plasma are not possible. Previously collected seminal plasma can be added to these doses (5-10 milliliter per dose) to compensate for the lack of seminal fluid in the collection. TD^NP Indicates that the collection is too dilute and although final AI doses will contain more than 10 percent seminal plasma, the final dilution rate will be less than 1 part semen to 5 parts extender. It is not recommended to store this semen for an extended time. Semen may be used immediately or for seminal plasma collection.

Table 3. The amount of extender (bold type) added (final dose volume in parentheses) to dilute boar collections for producing AI doses of semen containing 4.0 billion spermatozoa with at least 10 percent of the volume consisting of seminal plasma.^{a, b}
	Total Motile Sperm Cells in Ejaculate (1 x 10⁹) (Number of Potential AI Doses)
Ejaculate Volume	40 (10)	50 (13)	60 (15)	70 (18)	80 (20)	90 (23)	100 (25)	110 (28)	120 (30)
100	750 (85)	1005 (85)	1025 (75)	1070 (65)	TC^NP	TC^NP	TC^NP	TC^NP	TC^NP
150	850 (100)	1150 (100)	1125 (85)	1380 (85)	1450 (80)	1575 (75)	1475 (65)	TC^NP	TC^NP
200	TD^NP	1100 (100)	1400 (100)	1330 (85)	1500 (85)	1525 (75)	1550 (70)	2040 (80)	2050 (75)
250	TD^NP	TD^NP	1350 (100)	1550 (100)	1450 (85)	1705 (85)	1875 (85)	2130 (85)	2350 (85)
300	TD^NP	TD^NP	TD^NP	1500 (100)	1700 (100)	1655 (85)	1825 (85)	2280 (85)	2300 (85)
350	TD^NP	TD^NP	TD^NP	TD^NP	1650 (100)	1950 (100)	2150 (100)	2130 (85)	2250 (85)
400	TD^NP	TD^NP	TD^NP	TD^NP	TD^NP	1900 (100)	2100 (100)	2400 (100)	2600 (100)
450	TD^NP	TD^NP	TD^NP	TD^NP	TD^NP	TD^NP	2050 (100)	2350 (100)	2550 (100)
500	TD^NP	TD^NP	TD^NP	TD^NP	TD^NP	TD^NP	TD^NP	2300 (100)	2500 (100)
550	TD^NP	TD^NP	TD^NP	TD^NP	TD^NP	TD^NP	TD^NP	TD^NP	2450 (100)
^a Volumes are in milliliter. ^b 85-milliliter is considered an optimal dose volume whereas volumes below 60 milliliter are sub-optimal for sperm transport. TC^NP indicates that the collection is too concentrated to dilute for making AI doses at 4.0 billion per dose. Final AI doses of more than 60 milliliter with 10 percent seminal plasma are not possible. Previously collected seminal plasma can be added to these doses (5-10 milliliter per dose) to compensate for the lack of seminal fluid in the collection. TD^NP Indicates that the collection is too dilute and although final AI doses will contain more than 10 percent seminal plasma, the final dilution rate will be less than 1 part semen to 5 parts extender. It is not recommended to store this semen for an extended time. Semen may be used immediately or for seminal plasma collection.

Adding Seminal Plasma to AI Doses

Some boar collections consist of very low or high concentrations of sperm, so adjusting the dose volume is not possible. Therefore, when a boar produces a high volume of fluid containing few spermatozoa, consider collecting the seminal fluid by centrifugation and adding that seminal plasma to boar ejaculates that contain small amounts of fluid but large numbers of sperm. This can be accomplished by centrifugation for 10 to 20 minutes at 10,000 RPMs. Then remove the fluid portion, freeze if at -20^o C and thaw and use when needed. For example, a boar collection that consists of 100 milliliter of fluid containing 80 billion spermatozoa will produce 26 doses of semen (3 billion per dose). However, when diluted to a final dose of 60 milliliter (minimum needed for optimal sperm transport), the final dose only contains roughly 6 percent seminal fluid (100 ml ÷ 26 doses = 3.8 ml of raw semen per 60-milliliter dose). Because it is not advisable to lower the dose volume, adding 5 to 7 milliliter of pre-collected seminal plasma to the dose will adequately adjust the final percentage of seminal plasma to 15 percent. In some cases you will probably add only small amounts of seminal plasma, but this small amount may go a long way in terms of fertility.

Author's Note: Generally it is not recommended to fall below a 1:4 dilution rate (i.e. more than 40 milliliter of neat semen in a 100-milliliter AI dose) in order to maximize long-term storage of boar semen. Even though semen can probably be stored for up to 3 days at this rate, optimal long-term storage (depending on the boar) is more likely to occur at a higher dilution rate (i.e. 1:5 and higher).

Kevin Rozeboom

Suggested Readings

Rozeboom K. J., M. H. T. Troedsson, and B. G. Crabo. 1998. Characterization of the post-mating uterine inflammatory response in the gilt. J. Reprod Fertil. 114:195-199.

Rozeboom K. J., M. H. T. Troedsson, T. W. Molitor, and B. G. Crabo. 1999a. The Effect of spermatozoa and seminal plasma on leukocyte migration into the uterus of gilts. J. Anim. Sci. 77:

Rozeboom KJ, Troedsson MHT, Shurson GC, Hawton JD and Crabo BG. (1997) Late estrus or metestrus insemination subsequent to estrual inseminations decreased farrowing rate and litter size in swine. J. Anim. Sci. Journal of Animal Science 75 2323-2327

Soede, N. M., and B. Kemp. 1997. Expression of oestrus and timing of ovulation in pigs. J. Reprod. Fert. Suppl. 52:91-103.

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Last modified February, 15 2000.