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The development of modern long-life storage diluent for fresh ram spermatozoa - LK0661

Sheep breeding in the UK remains largely an extensive natural mating system involving small flocks with little biosecurity and disease control. The limited artificial insemination (AI) that takes place (about 50,000 inseminations per year) is of little impact, even though its exploitation enables farmers to benefit from genetic improvement programmes and to increase their biosecurity. The situation is in part due to technical limitations of the semen preservation methods currently available. Although semen freezing and laparoscopic AI procedures work well in sheep, the necessary technical expertise and attendant costs mitigate against its being extensively adopted; the development potential in AI depends on fresh semen inseminated cervically. The introduction of AI centres holding rams for semen collection, processing and distribution, along the lines of pig AI centres, would be of major benefit to farmers. This innovation has been recognised as being particularly suitable in the Welsh context where AI with fresh semen is growing in importance, and one company (CBS Ltd) has already established such an AI centre. However, it is also recognised as having great potential in England and Scotland.

The lack of suitable diluents for sperm storage imposes a major limitation with this exercise because, unlike the situation with cattle and pigs, there is currently no reliable extender for fresh ram semen that provides more than a few hours storage at ambient or chilling temperatures.
The proposal aims to solve this problem by developing a suitable diluent for longer-term semen storage.

Our previous work with pig spermatozoa has shown that the oviduct epithelium extends the life of spermatozoa in the female reproductive tract. So far, we have demonstrated that the apical membrane of the oviductal epithelium contains peripheral proteins that sustain spermatozoa in a live quiescent condition for up to 14 days. We are in the process of isolating and characterising these factors for possible inclusion in diluents for AI; this work is supported by a BBSRC LINK grant to the academic partners. We are also extending this work into cattle, and believe that the same approach would be applicable with sheep. Investigations of sperm-oviduct interactions therefore represent an important aspect of this application. We recognise, however, that the extensive literature on ram semen extenders contains reference to several formulations that are claimed to improve the shelf-life for ram semen. Nevertheless, their effectiveness under field conditions remains untested. There is considerable risk of economic loss if practitioners were suddenly to change extenders without the reassurance of field trial data, and for this reason we propose that this project should include both laboratory and field trials to address this issue. Such field trials are also necessary in order to establish the baseline (control value), against which any further beneficial effects of oviductal protein additives could be tested.

The potential benefits of this development are:
a) the ability to use disease-free pedigree rams with high genetic merit more widely (some 20 inseminations can be carried out with fresh semen from a single ram ejaculate) thus facilitating the rapid upgrading of the quality of the national flock,
b) reducing sheep movements and providing an improvement in biosecurity, and
c) progressing the National Scrapie Plan more efficiently.

This can only happen if cervical insemination programmes with fresh semen are made more efficient in terms of semen delivery since with current technology the costs would be prohibitive.
1. To identify from the published literature a group of 3-4 of the most promising ram sperm extenders, and to test the potential of the extenders (supplemented to control oxidative processes and membrane degeneration) under laboratory conditions to support extended functional sperm cell life for 24-48h at ambient or lower temperatures.
2. To establish that sheep oviduct epithelial cells and their components are capable of extending the lifespan of spermatozoa in vitro, under cell culture conditions at 39ºC.
3. To examine the ability of oviductal epithelial cell extracts when added to supplemented extenders to extend the functional life of sperm beyond 48h when stored at ambient or lower temperatures
4. To conduct field trials of promising diluent formulae to assess fertility under farm conditions. The existing commercial AI protocol would be used as the control in an initial field trial, and an extended storage period would be included in the trial designs.

The short-term, and minimum, outcome of this project would be: (1) the realistic identification of a ram semen extender that provides better fertility results than is currently attainable with current procedures; or (2) the demonstration that current protocols cannot be bettered using existing technology. This sound evidence-based information will be of real value to sheep breeders. The longer-term practical goal is to extend the lifespan of ram semen using the physiological mechanisms of the female reproductive tract as the basis for enhancing sperm survival.
Project Documents
• Executive Summary : LK0661 Executive Summary   (3401k)
Time-Scale and Cost
From: 2005

To: 2008

Cost: £490,835
Contractor / Funded Organisations
Britbreed Ltd, Zoological Society of London, IMV Technologies, University - Sheffield, Royal Veterinary College, CBS Technologies Ltd
Livestock Farming              
Sustainable Farming and Food              
Sustainable Production