Training farmers to perform artificial insemination in sheep

Summary

This two year project investigated a farmer-friendly, low cost artificial insemination (AI) technique for sheep.  The team consisted of four farmer producers from two farms, and two veterinarians, one of whom is also a producer, for a total of three farms.  Although laparoscopic AI (a surgical procedure) is becoming more common in sheep, the application of AI at the farmer level is limited.  A farmer-friendly technique such as vaginal AI would allow large segments of the sheep industry to make faster genetic progress similar to other livestock industries, with decreased biosecurity risk.

Introduction

AI in sheep is more complicated than other livestock species due to the anatomic complexity of the ewe’s cervix and the difficulty in preserving or extending fresh ram semen.  Semen extenders are used to dilute the semen, allowing it to effectively cover more ewes, and to provide nutrients for the sperm and protect against metabolic and cold shock, thus extending the viability of the semen. One of the challenges with sheep is that ram semen doesn’t freeze as well as other species, and fresh semen has proven difficult to extend beyond 24 hours in the past, making shipment of fresh semen difficult.  Extending the viability of fresh semen beyond 24 hours would allow more options for use.  

Laparoscopic AI is one solution that addresses both problems.  Because semen is deposited directly into the uterus, conception rates are higher, and frozen semen can be used.  But, laparoscopic AI is a surgical procedure, so it is expensive and requires a veterinarian.  Ewes are typically transported to a central location, further increasing costs and stress for the animal.

Vaginal AI was chosen for this project because it is noninvasive and safer for the ewe, easier for lay farmers and producers to perform, has low technical requirements and is more cost effective.  This procedure has decreased conception rates compared to laparoscopic AI, but the ease of accomplishing the procedure makes it an acceptable method. 

For this project, team members were trained on proper collection and handling of semen, as well as vaginal insemination techniques.  Conception rates were monitored over two years on all three farms. The team also evaluated recent research on ram semen extenders and analyzed several new extenders.

Objectives/Performance Targets

The objective of this project was to show that it is feasible to train shepherds to be proficient in performing posterior cervical (vaginal) AI, as well as collecting and handling semen.   Specific objectives included having the team members:

• Learn and gain experience in posterior cervical AI;
• Learn practical techniques for semen collection, evaluation, preparation and storage; and
• Use and compare the performance of three different semen extenders.

Methods

Each farm (Bielek, Duerr and Brown) supplied 20 ewes each year for this project.  All of the project ewes had at least one prior lambing and were in good health.  Ewes were randomly assigned to one of two farmer inseminators.  The inseminations were performed at the usual breeding time on each farm and the ewes were maintained on their home farms from AI through lambing. 

Estrous synchronization was used so that all ewes would come into estrous at the same time, allowing for timed AI.  This was necessary to accommodate the schedules of the entire team and to allow the use of freshly collected semen, which requires all the ewes to be in estrus at the same time. Estrous synchronization was achieved through the use of CIDRs, Estrumate and PG600.  CIDR is an intra-vaginally placed device that contains a natural progesterone hormone which is released at a controlled rate into the blood stream to control the ewe’s current cycle, allowing for accurate prediction of ovulation. The CIDR was inserted by the shepherds into their 20 ewes on each farm two weeks prior to breeding.  Injections of Estrumate and PG600 were administered when the CIDR was removed to stimulate ovulation. 

A breeding soundness exam (BSE) was done on each ram used for AI and included a physical examination and microscopic analysis of a semen sample to establish sperm volume, concentration, motility and morphology.  Any rams identified with questionable or unsatisfactory results were not used in the project.  In addition, on each farm, semen from three of the same rams used for AI was randomly selected for extender testing.

At approximately 35 days post insemination, Dr. Zimmerly, with team member assistance, performed transabdominal ultrasound to determine conception rates.  A natural service sire was used for any ewes not pregnant through AI.  Records were maintained of the ultrasound results and the actual number of lambs born, and alive at one week of age.   

The extenders we chose to analyze were selected based on recent research, advice from our advisors, and team members’ experiences.  Samples were evaluated to assess the percentage of progressively motile sperm cells, morphology and motility over time.  Evaluations were performed at 24 hour intervals until 20% motility had been reached. The time lapsed comparison of three extender formulas each year allowed us to identify the best formula for preserving sperm viability for our project.

Outcomes and Impacts 

Year 1

The group met at the Duerr farm on September 15, 2013 for training on BSE, semen collection and preparation and AI protocol.

On September 29 the team met at the Bielek farm.  Placement of CIDRs and injection of Estrumate and PG600 had been performed previously as outlined above. The ewes were sorted into desired breeding groups.  A BSE was performed on each ram and semen collected using an electro-ejaculator.  Each ram sample was evaluated immediately upon collection for volume, motility, concentration and morphology. These four factors dictated the number of doses available from that sample, using an extension ratio of 1: 3 (raw semen to extender) and an insemination dose volume of 1mL.  The ewes designated for each ram were inseminated immediately after the semen evaluation was completed and before collecting another ram.  To determine if there were differences between inseminators, two of the farmers, Anne Brown and Jeff Bielek, each inseminated half the ewes on each farm. At the Bielek farm in Year 1, however, each of the farmer team members was given the opportunity to inseminate at least one ewe.  Twenty ewes total were inseminated. 

Ten days later the ewes were put into breeding groups with the same rams they had been bred to using AI. The rams were fitted with marking harnesses.  Two weeks following the AI, ten of the ewes were marked by a ram, which suggested a 50% success rate.  Thirty five days following the AI, ultrasound was done on all the ewes.  These results were consistent with the natural service marking and also showed a 50% success rate.

The same procedures were followed on the Duerr farm on November 1 and on the Brown farm on November 30, with 20 ewes each, with similar success rates.  On the Duerr farm, we found that some rams did not respond to the electro-ejaculator, so an artificial vagina (AV) with jump ewe collection was attempted.  The volume of semen collected using the AV was higher, and only the AV method was used on the Brown farm.   

Semen samples were collected from three rams on each of the three participating farms for extender testing, for a total of nine per farm or 27 total evaluated samples. As mentioned above, collection procedures began with electro-ejaculation on the first farm, and proceeded to artificial vagina (AV) with jump ewe collection. Three doses were taken from each ram sample and chilled in three different extenders, then evaluated for vitality and motility at 24, 48 and 72hrs. A total of six different extenders were trialed, in order to identify which provided the greatest vitality and longevity. Extenders employed were a home-mixed UHT milk + egg potion and commercial Next Generation Universal semen extender base in plain, Amikacin + KPenn, and Timentin antibiotic (AB) variations. In addition, INRA-96 and Botu-semen were used on the Brown farm. On the first two farms, viability was found to be very low, regardless of extender or ram, suggesting a mechanical fault. In view of this, the handling protocol was adjusted for the third (Brown) farm. Samples were chilled and held in Exodus Equine Express II shipping containers. 

In view of the poor time-lapse chilled motility results in all samples on the first two farms, regardless of farm, ram or extender, the handling protocol was altered to address the following issues. Stored semen may be sensitive to the exposure to air in the 10mL tubes, so the third trial stored the semen in 1mL non-spermicidal syringes, with all air carefully expressed. Next, the Equine Express 11 shipping/holding containers are calibrated to chill a fluid volume of 80-100mL. Since all of the ovine samples totaled only 9mLs, the resulting altered chill rate may have caused excess sperm cell death. For this reason, two 40mL ballast syringes of sterile water were included in the chilling container to correct the chill factor. Finally, the coolant packs in the first two trials (Bielek and Duerr) were not changed during the 48hr time period, possibly allowing the samples to become too warm, elevating their metabolism and hastening death. For the third trial, the coolant pack was refreshed with a fully chilled pack at each 24hr evaluation period, maintaining the chill within original specifications. Results proved much greater longevity and viability on the third farm chill testing with the INRS-96 surviving > 50% up to 4 days and the Botu-semen surviving >50% for up to 3 days.

Year 2

Breeding soundness exams were performed on the rams to be used for AI at the Duerr and Bielek farms on August 23 and at the Brown farm on August 24, 2014, with satisfactory results on all farms.  The jump ewe with AV method was used on all farms, based on the success we had with this method in year 1.  The only concern with this method was that most rams are unaccustomed to having an audience during breeding and some were shy or reluctant to approach the ewe on a lead line.  Don and Anne Brown have extensive experience with AI in horses. At their suggestion, rams on some farms were “trained” in the collection process.  This training simply involved getting the rams used to approaching a ewe on a lead line and having people around when he mounted the ewe.  When done daily for several days before collection, this seemed to greatly increase the success.

The same timed AI protocol was used in year 2, with CIDRs inserted two weeks before breeding, and Estrumate and PG600 injections given when the CIDRs were removed.  AI was performed on the Bielek farm on October 4, which happened to be an unusually cold day.  One ram lamb took longer to collect than the others, and semen from this ram experienced some cold shock.  As in year 1, ewes were exposed naturally seven days post AI to the same rams they had been bred to using AI. The rams were fitted with marking harnesses.  Two weeks following the AI, 12 of the ewes were marked by a ram, which suggested a 40% success rate.  Thirty five days following the AI, ultrasounds were done on all the ewes.  These results were consistent with the natural service marking and also showed a 40% success rate overall, although the success rate for the semen with cold shock was only 14%, while the other two rams had a much higher success rate of 54%.   

The team met at the Duerr farm on November 22.  Semen had been collected from one of the Bieleks’ rams the day before, chilled and stored overnight to test how well storage in the chosen extender would work.  Due to an ice storm the morning of the 22^nd, the team met about three hours later than planned.  The stored semen was used first to inseminate six ewes.  We then attempted to collect two additional rams, with no success, first with jump ewe/AV method, then using an electro-ejaculator.  After several attempts using both methods on both rams, a decision was made to expose the remainder of the flock naturally to the Polypay ram.  The ram was fitted with a marking harness and released with the remaining 26 ewes at 4:00 pm while the ewes were still in estrous and 54 hours after the CIDRs were pulled.  All but eight of the ewes were covered within 72 hours.  The breeding harness color was changed at two weeks and the remaining eight ewes were covered.  Ultrasound was performed on January 4, 2015 on the six ewes bred via AI with four of the six scanning as pregnant, a 67% success rate.  Ultrasound was not performed on the remaining ewes, but none were marked by a cleanup ram, so were assumed to have all settled.

The team met at the Brown farm on November 28.  All the ewes had been placed in a pasture next to the three rams for two weeks prior to the insemination date. On the morning of November 28, ewes were all displaying behavior consistent with standing heat, and all three rams were responding with aggressive mating behavior. AI day was very cold, and maintaining the semen handling equipment temperature at or near 100 F was difficult. Collection procedures began mid-afternoon. All three rams displayed great willingness to tease/mount ewes in chute, and appeared to climax to ejaculation in the AV, however no fluid ejaculate was obtained. Each ram produced only 1-2 mL of froth, which, upon microscopic examination, contained no viable sperm cells. This may have been caused by the cold temperature and difficulty attaining proper heat and pressure in the AV. After a minimum of four attempts per ram with no successful ejaculate, ewes were divided and turned out with their designated rams for natural cover. No AI was able to be performed.  Since fine wool is a product on this farm, marking harnesses were not used, but copulatory behavior was observed immediately by every ram upon turnout with ewes.

Ultrasound examination of all 29 ewes was performed on January 6, 2015, at 37 days post initial exposure to rams, with16 of 29 ewes ultrasounded pregnant from first ovulation.  This computes to a 55% conception on first ovulation, via natural cover, with the CIDR synchronization protocol.  Any ewes that scanned open with ultrasound were turned back out with their respective rams for one more cycle, or until January 15, 2015. Ultrasound was performed again on March 12, 2015 to determine overall conception from later ovulations.

Accomplishments

Overall, each member of the team learned to synchronize estrous using CIDRS, and to inseminate ewes using vaginal AI technique.  We found that training the rams, or getting rams accustomed to handling during breeding, helped improve the success rate of semen collection using an artificial vagina.  Our results in the second year were greatly hindered by our difficulties in semen collection. 

The average conception rate across all three farms in Year 1 was 38%, with a range of 20% to 50%.  In Year 2, the average conception rate for the ewes actually inseminated was 35%; for ewes inseminated with fresh semen the rate was 40%.   The table below provides a summary.

Potential Contributions

Vaginal AI was shown to be an acceptable method for producers to economically artificially inseminate their sheep flocks.  However, some technical assistance would be needed for most farmer producers in order to achieve a good outcome. This project helped to underscore the importance of breeding soundness exams in rams.  We feel that further training and research pertaining to semen collection and handling are needed before this method will receive wide-spread adoption.

Publications/Outreach 

In Year 1, we partnered with the Ohio Heartland Sheep Improvement Association (two of our team are members of OHSIA) to co-sponsor their annual fall educational workshop on September 28, 2013.  The focus of the workshop was breeding and genetics, and three members of our group were speakers: Dr. Duerr, Dr. Zimmerly and Kathy Bielek.  Information presented included biosecurity, breeding soundness exams, the benefits and challenges of AI, genetic selection and a BSE demonstration.

Social media was used to not only raise awareness but also to provide outreach for our project.  A blog, web site, and Facebook page was created entitled ‘Breeding Better Sheep’.  The team felt it important for online project specific posts to lag behind actual events as we digested the information we were learning ourselves.   Therefore, in order to help build an audience in 2013, articles related to sheep were shared with our Facebook Fans two to three times a week.  In the last quarter of 2013, the Facebook page was promoted to various sheep organizations on a local, regional and national level.  At the beginning of 2014 the page had 235 fans and each post reached an average of 50 people.  Unfortunately, Ginger Davidson, the team member responsible for the social media portion of the project had to withdraw from the project for health reasons and did not participate in year 2 of the project. 

Two members of this proposal, Don Brown and Dr. Craig Zimmerly, were asked to give a presentation of this work at the 2014 and 2015 Ohio Ecological Food and Farm Association (OEFFA) annual conference in February, 2014 and 2015. Copy and paste this link to view a video of their presentation: https://youtu.be/91tKLGoP9ys?list=PLQLK9r1ZBhhGr9RLwfvRvJLEtHJOLdXZz

A workshop was given in November, 2014 on an Amish farm in Sugarcreek, Ohio to share our results with the local Amish community.  This workshop resulted in a detailed write-up of our project in the Farm and Dairy newspaper.

All members of the team are very active in the sheep industry and continue to share information about the grant on an informal basis with their many contacts.

Future Recommendations

We need new/more effective means for disseminating information. We also need to promote BSE to ram owners, as well as the potential market for shipped semen, which may increase the value of their rams.