Genetics/Reproduction

Handling Affects Fertility of Frozen Semen

Storage of liquid boar semen for one to five days at 15-17°C. (59-63°F.) in commercial extenders has accelerated the use of artificial insemination in the U.S. swine industry the past 10 years.

Popular extenders, such as Beltsville Thawing Solution (BTS), MRA and Androhep Plus, are also being used to "slow cool" boar semen to 15°C. in preparation for cryopreservation.

Typically, when boar semen is being readied for freezing in straws, the extended semen is held for three hours at 15°C. However, in some situations, this standard protocol must be altered to accommodate shipping of boar semen from its collection point to another location for preparation, freezing and storage for use later.

The need to ensure maximum sperm survivability is a higher priority since the USDA National Animal Germplasm Program began receiving boar semen for cryopreservation to protect and maintain genetic diversity, and to serve as a genetic bank in case of a major animal germplasm loss through disease, natural disaster or terrorist attack.

Cryopreserved boar semen is already at a disadvantage compared to liquid fresh semen, with respect to farrowing rate and litter size. For those sending semen to another location or holding it overnight before cryopreservation, sperm-fertilizing capacity could be decreased further. Therefore, a greater number of thawed sperm may be required to make up the difference.

For these reasons, an experiment was conducted to compare the effect on semen characteristics and fertility after freezing sperm on the day of collection and one day after collection.

One ejaculate from each of six boars was held in either BTS or Androhep Plus for either 3 hours or 24 hours at 15°C. to simulate overnight shipment, then frozen in 5 ml. straws.

The spermatozoa were thawed and 31 estrus-synchronized gilts and sows were inseminated approximately four hours before the expected time of ovulation, induced by injection of 750 HCG (human chorionic gonadotropin).

The pregnancy rate 23 days after insemination favored the semen held for three hours compared to the 24-hour holding time — 75% vs. 53%, respectively, although the difference was not statistically significant. However, embryo number/female and percent embryo survival were decreased from 15 to nine and from 73% to 46%, respectively, when holding time at 15°C. was increased from three hours to 24 hours.

Pre-freeze and post-thaw sperm motility, plasma membrane integrity, and acrosome morphology were of little value in predicting the decrease in sperm fertility after increasing holding time from three hours to 24 hours.

These results are important because they indicate that fertility of thawed sperm will be reduced after overnight shipping of liquid semen to another location for cryopreservation. The same sperm would likely remain fertile if shipped in liquid form.

More work is necessary to determine the molecular mechanism by which cryopreserved sperm lose fertilizing capacity if they are not frozen on the day of collection.

Researcher: H. David Guthrie, Beltsville Agriculture Research Center, Beltsville, MD; phone 301-504-9020 or e-mail [email protected]

Swine often exhibit high rates of early embryonic mortality (>30%), and the efficiency of producing swine embryos using in vitro fertilization and embryo culture methods is poor compared to other major livestock species. At best, 20% of transferred invitro produced embryos develop into living offspring after embryo transfer.

The maternal recognition of pregnancy combined with the dramatic embryo elongation that occurs between Day 11 and Day 12 of gestation are especially critical stages in pig development. It is between Day 11 and Day 12 that embryos are highly susceptible to early embryonic loss. Researchers believe that identifying the genes that are switched on and off during this interval are a critical step in developing new tools and strategies for decreasing embryonic mortality (Figure 1).

This study represents the first successful application of the new technique, Serial Analysis of Gene Expression (SAGE), in livestock embryo research.

Researchers from USDA Agricultural Research Service Germplasm and Gamete Physiology Laboratory are pioneering SAGE to discover and study the genes that are differentially expressed during the critical stages of early embryonic development. The SAGE technique enables them to capture a gene expression profile, or snapshot, of gene activity at specific stages of embryo development. These gene expression profiles are comprised of a short "SAGE tag" of DNA nucleotide sequence information allowing each respective gene to be identified along with the frequency with which each "tag" occurs.

Researchers recently completed a comparison of SAGE tag profiles comprised of over 43,000 SAGE tags obtained from Day 11 to Day 12 swine embryos. Approximately 246 potentially different genes were expressed at significantly higher frequencies (135 tags) or lower frequencies (111 tags) between Day 11- and Day 12-stage embryos.

Nucleotide sequence comparisons between the pig embryo SAGE tags and public DNA sequence databases indicated that the 54 most different tags were clustered in the following commonly accepted functional groupings: eight metabolic enzymes, three heat shock proteins, three transcription factors, 19 hypothetical genes or proteins, and 21 unknown or novel genes. Follow-up experiments are being conducted to confirm and verify the role these SAGE gene tags play in pig embryo development.

The results of the present and ongoing SAGE research will ultimately identify the genes critical for swine embryo development. These genes could then serve as powerful tools for developing new strategies to lower early embryonic mortality.

For pork producers, in addition to reducing embryonic losses in the early stages of gestation, there is an additional potential benefit of increasing rates of genetic improvement through more extensive integration of maternal genetics in breeding programs, through improved and more efficient methods for producing swine embryos in vitro. Click here to download Figure 1. (This requires Adobe Acrobat Reader, download at: www.adobe.com.)

Researcher: Kurt A. Zuelke, DVM, USDA Agricultural Research Service, Beltsville, MD; phone 301-504-8545 or e-mail [email protected]

Improving overall herd reproductive performance by enhancing semen uptake has been attempted by adding hormones to semen at the time of artificial insemination (AI).

University of Illinois researchers investigated the effects of adding oxytocin (0.2 cc.), prostaglandin (1 cc.), estrogens (˜11 mg.), or no hormone (control) into semen immediately prior to AI.

To determine the impact of hormone addition, the researchers utilized a sensitive, low fertility model that was based on a single insemination of only a half a billion sperm in 80 cc. (volume). This low fertility model would simulate field cases of infertility and thereby allow detection of subtle differences in semen volume backflow and sperm loss after AI. Researchers also determined the impact on uterine contractions and effect on number of sperm in the female tract at eight hours following AI. Numbers of fetuses and pregnancy rate at Day 45 of gestation were also assessed in gilts.

The addition of hormones to semen did influence the pattern of backflow over an 8-hour period following AI, but did not influence the total volume lost at the end of this period (85% of volume) when compared to controls (90% of volume). This pattern was similar for the amount of sperm lost over the eight hours, which was also altered by hormone addition to semen. Again, the total percentage of sperm lost at the end of eight hours was not different (38%) compared to controls (54%), as shown in Figure 1. (This requires Adobe Acrobat Reader, download at: www.adobe.com.)

The addition of hormones to semen tended to have a positive effect on the numbers of sperm found at the tip of the uterus at eight hours following AI when compared to controls (60,000 vs. 22,000). However, this increase was not evident in the oviducts.

Within a 2-hour period following insemination, the frequency of uterine contractions was only increased at 30 minutes after insemination with prostaglandin. The frequency, amplitude and duration of uterine contractions were not affected at any other time by any other hormone treatment.

The pregnancy rate for this low fertility model was 59%, and was not affected by the addition of hormones. Total number of healthy fetuses was increased with prostaglandin and tended to be higher with oxytocin when compared to controls (Figure 2).

Researchers suggest that in cases of lowered fertility, these levels of hormones induce alterations in volume and sperm backflow patterns and tend to increase uterine sperm numbers, which translates into more, healthier fetuses.

In cases of low fertility, such as where poor quality semen and/or poor inseminations occur, when hot weather is a factor and Parity 1 or 2 sows are being mated, hormone addition to semen could reduce the risk of infertility.

Researchers: K. Willenburg, S. Rodri-guez-Zas, G. Miller, and R. Knox, University of Illinois. Phone Knox at (217) 244-5177 or e-mail [email protected]