Cervical insemination: the method of the future


Artificial insemination offers significant advantages to livestock producers however today the only commercially viable assisted reproductive technique to achieve pregnancy in small ruminants is via invasive laparoscopy requiring full sedation of the ewe and surgical invasion. Unfortunately, small ruminants are highly susceptible to risk of death from the procedure [1], run the risk of infection, and requires a trained 3rd party and veterinary assistance, greatly increasing expense. Cervical insemination, commonly used in cattle, is a revolutionizing technology to increase genetic gain and decrease risks associated with male animals, decrease expense (>50%), allow greater income per animals (est >100%), and improves animal welfare but is difficult in sheep because of their particular cervical conformation; ewes have between 2 and 7 rings [2,3] that are often offset from one another. Membrane Protective Technologies Inc (MPTI) is developing a new, all-natural technology in assisted reproductive technologies in the sheep industry by enabling cervical insemination.

The long term objective of MPTI's project is to develop a full system that enables the use of cervical artifical insemination in sheep by the trained lay person. The development of this technology is multifaceted and includes optimized unique extenders and sperm handling techniques, cryopreservation methods, as well as sperm delivery techniques, and possibly insemination devices. In previous work, MPTI developed sperm related cryopreservation technology and this will be applied to the second objective.
Within this grant proposal MPTI will focus on the key aspect of insemination, the consistent passing of an AI rod into the cervix via development of a cervical ripening agent for ewes. Such an agent assists in insertion of AI rods into the reproductive tract by slightly, and temporarily, modifying the physiology of the cervix. Success will be determined by an agent that enables the passing of an AI rod through at least 1 of the cervical rings in 80% of the treated ewes. Secondarily, a 35 day pregnancy rate of at least 40% in treated ewes will indicate success of the preliminary system.

MPTI has partnered with Crego Livestock, a prominant and long time club lamb producer. This partnership ensures technology developed will be commercially relevant and allows rapid dissemination to the industry. Crego Livestock will continue to assist in acquisition, boarding and care of sheep as well as commercial guidance, during all project-related experiments.

RESEARCH BACKGROUND: Preliminary research using abattior reproductive tracts was used to limit the cervical ripening agents from a field of 10 to three, plus a positive control that worked consistently. For proprietary reasons, the three agents are not being disclosed but are all natural, plant derived extracts that are physiologically compatible with the delicate cervical tissues. Newly acquired abattior tissue responds similar to live tissue for a period of time post mortem.

ANIMALS: To assess effectiveness of the cervical ripening agents in situ, thirty-five (35) multiparous ewes will be acquired. Ewes will be placed on a feeding program to bring the animals to ideal body condition (score of 2.5-3 out of 4) for breeding and pregnancy. Thirty ewes of the best quality will be synchronized using a standard 11 day synchronization protocol [9]. Assuming a response rate of >80%, 24 or more ewes determined to be in estrus via response to a programmed wether, will be divided into 4 treatment groups of 6 ewes each. Ewes will be divided to a balance of breeds, ages, and parity.

EXPERIMENTAL TREATMENT: Before treatment, cervical tone will be measured using the technique of Perry [4]. Briefly, a a 0.25cc AI rod will be passed into each ewe. A mark will be made on the rod outside the ewe (where the rod disappears into the vulva) when it reaches the external cervical os. Then a second mark in a different color will be made at the point the rod cannot be passed further without manipulation and damage to the cervix, the rod removed then 15 mls of one of three cervical ripening agents will then be delivered via individual swabs, and allowed to incubate for 30 min. The same AI rod will be passed again. The distance between the two marks will be measured in centimeters as an indication of the depth traveled. Number of rings passed will be noted and time it takes the inseminator (single inseminator for experiment) to pass. Four replicates of each the 4 treatments (Agent A, Agent B, Agent C, negative control [sterile saline]) will be applied to the same set of ewes. Note, previous positive control was a prostaglandin which will not be used in vivo.
It is not economically feasible within the constraints of the budget to purchase, house and condition a sufficient number of ewes to treat one ewe one time. Therefore each set of ewes will be treated with the four different agents/treatment in sequence. Four synchronization will be applied to each ewe over the course of 7 months (three month without synchronization; see figure in supplement). This is a slight, but calculated risk as the ewes may develop cervical issues or become cystic. Extra ewes will be added in to the protocol if a ewe is unable to continue through all treatments.

INSEMINATION: During the last synchronization protocol and treatment, each of the ewes in this study will be inseminated using previously cryopreserved ram sperm. Sperm will be packaged at 75x10e6 sperm/dose using MPTI's previously developed GameteGuard®methdology and extender. Sperm will be deposited within the cervix or at the furthest point the AI rod is able to be inserted. Location of deposition will be noted and pregnancy per deposition and impact of location calculated both within and between treatments. Pregnancy will be assessed using transabdominal ultrasound 35-45 days post insemination, and confirmed by presence of an active heart beat.
Objectives of experiment are 1) cervical relaxation enabling passing an AI rod through more than one cervical ring, 2) enablement of cervial AI as identified by pregnancy confirmation. Number of required ewes per treatment was determined using a Lenth's power test (alpha=0.5, power=0.9). All data will be analyzed using a liner mixed model with replicate and ewe as random effects and treatment as a fixed effect. It is anticipated the 4 sessions will be completed in 7 calendar months of work time (calendar month does not equal treatment month; see timeline in supplement).
Measurements of success are 1) improvement in cervical penetration depth of 30% before versus after treatment, and 2) conception rate of 40% when sperm is deposited in an intracervical location. It is anticipated that treated cervices will relax more than non-treated cervices, allowing an insemination rod to progress further through a ewe cervix than those not treated. It is also anticipated that ewes inseminated deeper within the cervix will have higher 35 day pregnancy rates.