OPTIMIZING THE PROCESSING AND STORAGE OF LIQUID SEMEN IN HAIR SHEEP RAMS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: EVANS-ALLEN
• Reporting Frequency: Annual
• Accession No.: 1017943
• Project Start Date: Oct 15, 2018
• Project End Date: Oct 31, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
VIRGINIA STATE UNIVERSITY
(N/A)
PETERSBURG,VA 23803

Performing Department
Agriculture

Non Technical Summary
Artificial insemination (AI) in sheep has not evolved at a rate common in other types of livestock species. A major constraint in sheep is the inability to readily pass the cervix in ewes with an insemination gun. This greatly limits the use of frozen-thawed semen that should be deposited in the uterus to achieve acceptable pregnancy rates. Recently liquid semen AI system have received increased attention in sheep. In Europe and South America localized co-ops and networks have developed based on the use of short-term stored liquid semen and vaginal AI. Such systems have not been evaluated to any extent in the U.S., but could be of great benefit, especially to smaller flocks, to introduce unique and superior germplasm without the need for live transport of the animals and the associated problems with animal stress and disease transmission. Research in our lab has shown that pregnancy rates around 40% can be routinely achieved in hair sheep with using vaginal AI and semen stored in a simple skim milk-egg yolk extender within 12 hours of collection/processing. This project will evaluate if liquid-stored semen quality can be improved with the use of more complex extenders and better processing conditions. Experiments will be conducted to refine extender composition (egg yolk inclusion levels, addition of energy substrates and antioxidants), and processing and storage protocols (cooling rates, storage temperature, shipping environment). Results will help improve pregnancy outcomes in liquid semen AI systems and steps will be taken to improve adoption of this technique by the sheep industry.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3610	1020	100%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3610 - Sheep, live animal;

Field Of Science
1020 - Physiology;

Keywords

hair sheep

liquid semen

artificial insemination

Goals / Objectives
Any expansion of the sheep industry in the U.S. will be associated with smaller farm flocks, managed under forage-based production systems that can tap into local, high-value niche markets. For these farms to be viable they have to utilize sheep breeds and production systems that are profitable under the prevailing environmental conditions. A constraint for these small farms is limiting inbreeding, while maintaining a closed flock that benefits herd health. The availability of off-farm genetic material in the form of semen will greatly improve effective breeding management.This project expands on earlier work that evaluated the use of a simple liquid semen AI system for use on small, limited-resource farms. These earlier efforts were aimed at keeping inputs simple, and farmer expertise to a minimum. In this system, liquid semen AI pregnancy rates of approximately 40% could be achieved in commercial on-farm AI trials within a 3-hour drive from the collection and processing site. Further improvements in liquid semen processing and storage should improve these rates and will be evaluated in this project. In the absence of flocks with sufficient numbers of ewes to allow for extensive in vivo testing of techniques and protocols, in vitro measures of sperm motility parameters and sperm viability will be used to assess differences in treatment outcomes.The proposed project addresses the following objectives related to refining liquid semen AI in hair sheep:evaluate effects of extender composition on sperm motility and viability during cooled storage of liquid ram semendetermine optimum storage and shipping environments for liquid-stored ram semen as measured in sperm motility and viability

Project Methods
Semen samples for all experiments will be collected from Barbados Blackbelly and St. Croix rams maintained in a flock on Randolph Farm. Rams are managed as a single group in dry lot and are provided mixed grass hay and a corn/soybean meal supplement (14% crude protein) at 1% of body weight throughout the year. Rams are trained in the use of an artificial vagina, and samples are collected with aide of an estrus-induced teaser ewe.For all experiments in this project, semen samples from 8 to 12 rams will be pooled to eliminate effect of individual ram in the analysis model. Only ejaculates with a volume >0.5 mL and >70% motile sperm (subjective animal-side evaluation) will be used in the creation of semen pools. Concentration of individual ejaculates will be determined using a Minitube® SpermaCue spectrophotometer calibrated for ram sperm. Samples will be maintained in a water bath at 27°C at initial evaluation, and are processed and extended at this temperature. Semen will be extended to a final concentration of 250 x 106 sperm/ml, and loaded into 0.5 ml straws for use. For experiments, straws will be analyzed in triplicate.Sperm motility and motion characteristics will be determined with a computer-assisted sperm analyzer (CASA; Minitube© Androvison), and sperm viability by propidium iodine staining (Chemometec Nucleo counter SP-100).Objective 1:The initial experiment will refine our current protocol to assess sperm stress associated with maintenance at body temperate for 6 hours, simulating a post insemination environment. At five periods (0, 12, 24, 48, and 72 hours) of cooled storage (4°C) in our standard extender (5% v/v egg yolk in UHT skim milk), samples will be moved to into a CO2 incubator (at 39°C), and motility and viability recorded at 3, 6, 9 and 12 hours of incubation. The incubation period associated with a substantial decline in motility and viability will be used in subsequent studies as an indicator for decrease in sperm fertilizing capacity.The second experiment will evaluate the optimum egg yolk inclusion level for a standard liquid storage extender. Pooled semen will be diluted to 250 million sperm/ml in UHT skim milk extenders with inclusions of 0, 5, 10, 15, 20, and 25% of egg yolk. Samples will be evaluated after 24, 48 and 72 hours of cooled storage (4°C), and after incubation according to a time derived in the first experiment. Egg yolk concentrations with highest motility and viability will be used in the default extender for subsequent studies.Subsequent extender composition experiments will evaluate the effect of inclusion of trehalose, catalase, and methionine in the extender to provide additional protection for sperm membrane against low temperatures. A standard extender with optimal egg yolk concentration from the earlier trial will be used with or without the addition of 50 mM trehalose and 200 U/mL catalase and 1 mM methionine. Semen will be processed and analyzed for motility and viability as described above. This experiment will be set up in a factorial design to detect possible interactions between extender components and their concentrations.Objective 2:The initial experiment will evaluate storage water volume on cooling profiles of semen straws. For standard processing in the past we have been placing straws in 500 ml volumes water for cooling from processing to storage temperatures. In this experiment, straws will be placed in water volumes of 100, 500 and 1000 ml for cooling in the refrigerator (from 27 to 4°C). Cooling profiles will be determined at the straw level, and the effect of cooling profile on sperm motility and viability will be determined after 48 hours of storage.The second experiment will evaluate the feasibility of storage at a higher temperature (15°C) compared to our current standard of 4°C storage. After standard processing, samples will be placed in water baths maintained at either 4 or 15°C, and stored for a period of 96 hours. Samples will be removed at 24 hour intervals and motility and viability assessed. Cooling profiles will be recorded as straws are cooled to holding temperatures.The third experiment under Objective 2 will evaluate portable storage options for transport of cooled samples in a field environment. Samples will again be prepared using a standard protocol derived from the earlier experiments. Control samples will be stored in a water bath maintained at 4°C. Treatment samples will be placed either in a styrofoam box (5.5 L interior volume and 38 mm wall thickness) with blue ice cooler packs, a commercial stallion semen shipper (Equitainer®, Hamilton), or a portable 12V/24V refrigerator/freezer (AccuCool®). Temperature loggers will be placed with the semen straws in each container, cooling profiles and stability holding temperatures will be recorded. Samples will be evaluated at 24 hour intervals for a period 96 hours.Evaluation Plans:The work performed under this project will be subject to a critical quarterly review by research and extension cooperators. Criteria evaluated will be (1) the adherence to the proposed timetable, (2) the soundness of the data generated to this point, and (3) the solution to problems associated with animal management, and sample processing and analysis. Progress in data analysis and preparation of manuscripts also will be evaluated. Quality of the work performed will be assessed via acceptance of abstracts for presentation at society conference and publication in proceedings as well as acceptance of manuscripts in reputable journals in the field. Quality and relevance of producer workshops and field will be assessed through evaluation instruments provided to participants at these events.Dissemination Plans:The results obtained in this study will be prepared for presentation at scientific meetings, such as the annual meeting of the American Society of Animal Science and, and technical meetings of regional research projects (SSC-81: Sustainable Small Ruminant Production in the Southeastern U.S.; NCERA-214: Increased Efficiency of Sheep Production). Scientific manuscripts will be submitted to peer-reviewed journals covering applied animal science.Selected data from the project will be summarized in fact sheets that provide comprehensive, step-by-step descriptions for applying techniques on the farm level. These fact sheets will be made available both in printed form, as well as electronic media on a designated web site. On-line training will include streaming video. Results will also be made accessible to the industry through communications in sheep industry publications. These dissemination methods will help to ensure that information about the new procedures reaches small farms and rare-breed producers most able to benefit from the research.

Progress 10/15/18 to 10/31/21

Outputs
Target Audience:Animal science community, sheep producers, extension agents/specialists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training in field research technique, data collection and analysis, and preparation and presentation of results was provided to three undergraduate students. Hands-on on training of vaginal artificial insemination was provided to a group of sheep producers as part of a on-farm research/demonstration trial. How have the results been disseminated to communities of interest?Results were presented a several scientific conferences and as part of the station report in two regiona research projects (NCERA 214; SCC-81). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A series of experiments was conducted to evaluate optimal storage conditions for liquid-stored ram semen using sperm motion analysis as a measure of sperm quality. Furthermore an on-farm trial was conducted to evaluate the effect of semen dose on pregnancy rates using liquid semen vaginal AI under commercial conditions. Early experiments under this project have shown that temperatures of 5° and 10°C were suitable for liquid storage of ram semen, but no information was available on the effect of cooling rate. This expereiment evaluated motility characteristics of ram semen stored at 5° and 10°C in a liquid (refrigerated water bath) or air (variable temperature portable refrigerator) environment for 96 h. Two ejaculates were collected from 6 hair sheep rams using an artificial vagina, pooled and evaluated for initial motility and concentration. Samples were extended in ultra-heat treated skim milk and egg yolk (10% v/v) to a concentration of 250 million sperm/ml, and packaged in 0.5 ml straws. Semen was held at 32°C during processing and straws were placed in 500 ml jars for storage. Samples were removed from storage at 24 h intervals, and motility characteristics were determined using a computer-assisted sperm analyzer. Time to designated storage temperature was shorter in the liquid environment (5°C: 275 minutes; 10°C: 75 minutes) than the air environment (5°C: 500 minutes; 10°C: 654 minutes). Storage environment and temperature had no effect on sperm motility characteristics. Length of storage generally had no effect on motion characteristics (mean total motility: 44.1%), but slow and circular motility were affected. Results indicate that cooling rate did not impact sperm motion characteristics in liquid-stored ram semen. A second trial evaluated suitable shipping containers for liquid stored ram semen, as no commercial products are available as are for other species (equine, porcine, canine). The experiment evaluated a professional shipping device used for horse semen (Equitainer®), a commercial 10 L Igloo® cooler, and a generic 6 L styrofoam cooler for simulated shipping liquid ram semen. Cooling was generated using a custom coolant can, freeze blocks, and gel packs for equitainer, igloo cooler and Styrofoam box, respectively, and temperature was monitored using Hobo® temperature loggers. Semen was collected from 8 hair sheep rams using an artificial vagina, extended in UHT skim with 10% egg yolk to 250 million sperm/ml, and packaged in 0.5 ml straws. Straws were stored in 500 ml water-filled jars, and placed in the charged shipping containers. Straws were removed for analysis at 0, 6, 24, and 48 hours, and sperm motion characteristics evaluated using a computer-assisted sperm analyzer. Cooling rate was slower and holding temperature higher in the Equitainer (0.034°C/min; 14.4°C), but similar in the Igloo and Styrofoam containers (0.107 - 0.143°C/min, 2.32 - 2.93°C). After 48 hours storage progressive and rapid motility was similar in the Igloo and Styrofoam containers (60.2 - 63.8%, and 27.6 - 28.6%), but lower in Equitainer (32.9%; 14.4%). Results demonstrate that shippers designed for other species may not be suitable for rams, but that commercial coolers may be adapted for such use. In a third experiment Barbados Blackbelly hair sheep ewes on a commercial farm (n=92) were used to evaluate to further evaluate the use of liquid semen AI under commercial conditions. More specifically, the effect of semen dose on vaginal AI pregnancy rates was examined. Estrus was synchronized by inserting new and once-used vaginal progesterone implants (CIDR devices) for 8 days. Ewes were assigned to treatment after balancing by age to two groups, and inseminated once with either 75 (LOW), or 150 (HIGH) million sperm at 48h after CIDR removal by vaginal AI. Semen was collected on the day of insemination from Blackbelly rams (n=4) by artificial vagina, and ejaculates pooled. Subsamples of the pooled ejaculate were extended in an ultra-heat treated skim milk and egg yolk (10% v/v) extender to final concentrations then packaged in 0.5 ml straws. Semen straws were placed in a portable refrigerator and allowed to slowly cool to 5° C during transport to the farm (45 minutes). Sperm motility and concentrations in semen straws were monitored with a computer-assisted sperm analyzer (CASA) for 24 hrs. Pregnancy rates from AI was determined by transrectal ultrasound 21 d after insemination. Pregnancy rates tended to be greater in HIGH (37.8%) compared to LOW (21.3%), while CIDR type had no effect on overall pregnancy rate (29.3%). Sperm motility was not affected by sperm concentration or time of storage and remained stable until 24 hours of storage. In conclusion, higher concentrations of semen tended to increase pregnancy rates when ewes were inseminated, and results were in line with observations of earlier trials (average pregnancy rates of 40%).

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Robertson, M. O'Brien, D., Wildeus, S., 2022. Effect of semen dose on pregnancy rate using liquid semen AI in hair sheep. Proceedings, ARD Research Symposium, Atlanta, GA, April 4, 2022
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Brown, K., Wildeus, S. O'Brien, D., 2022. Effect of cooling rate on sperm motion characteristics of ram semen during liquid storage at two temperatures. Proceedings, ARD Research Symposium, Atlanta, GA, April 4, 2022
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Griffin, C., Wildeus, S. O'Brien, D., 2022. Retention of ram sperm motility during liquid storage in different types of shipping containers. Proceedings, ARD Research Symposium, Atlanta, GA, April 4, 2022

Progress 10/01/19 to 09/30/20

Outputs
Target Audience: Nothing Reported Changes/Problems:Active research on this project was suspended due to COVID. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Conduct trials on the effect of storage environment (cooling temperature and cooling rate) on sperm motions characteristics as a measure of sperm quality.

Impacts
What was accomplished under these goals? Due to the COVID pandemic no trials were conducted in the project during this reporting period.

Publications

Progress 10/15/18 to 09/30/19

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Experiential learning was provided to 4 undergraduate student workers as part of this project.A student also prepared an abstract and presented data from one of the experiments at a scientific conference. Work under this project also provided training opportunities for junior/senior level high school students enrolled in veterinary science curricula at regional technical centers. How have the results been disseminated to communities of interest?Results have been presented at two scientific conferences, and also as part of station reports at two small ruminant regional research project meetings. What do you plan to do during the next reporting period to accomplish the goals?Future research will look at the effect of liquid semenstorage temperature and cooling rates on semen quality characteristics. Furthermore, the use ofcommercially available custom shipping containers available for industries with established liquid semen artificial insemination protocols (horse and swine) should be evaluated.

Impacts
What was accomplished under these goals? Artificial insemination is not commonly practiced in small sheep farm settings due to low success rates using frozen semen, semen availability, and the high levels expertise and costs associated with itsapplication. Alternative techniques, such as use of liquid semen and simple vaginal semen deposition,are beingevaluated to make its use feasible on small farms. Our lab has developed a simple technique for vaginal artificial insemination using short duration storage of cooled semen. However, there is a need to optimize semen extender composition and storage environment for this technique. An initial experiment evaluated the inclusion level of egg yolk (0, 5, 10, 15, 20, and 25%) in a simple ultra-heat treated skim milk extenderon sperm motion characteristics during 96 hour storage at 5°C. Sperm motion charcateristics were determined using a computer-assisted sperm analyzer. Egg yolk inclusion at 10-15% achieved the highest level of progressive motility and fasted sperm forward velocity after 4 days of cooled storage. These egg yolk levels are higher than the 5 to 10%that had previously been used for liquid ram semen storage.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Mezil-Thomas, J., Wildeus, S. 2019. Rate of egg-yolk inclusion in a milk extender on sperm kinematics of ram semen during chilled liquid storage. Proc. ARD 19th Biannual Research Symposium, p. 63.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wildeus, S., OBrien, D. (2019) sperm motion characteristics of ram semen liquid-stored in a milk-egg yolk extender at four temperatures. J. Anim. Sci., Vol. 97, Suppl. 1. 78