VEIN : Sheep Health & Production : Chapter 8. Controlled breeding; artificial insemination (ai) and moet

This second chapter on reproduction is concerned with various direct interventions to manipulate and enhance reproductive function in sheep flocks. These procedures should be used only on healthy and well managed sheep. Flocks which are malnourished, suffering from parasites or other diseases or otherwise mismanaged are not suitable. The procedures usually involve considerable cost to your client, and their use should not be lightly recommended.

Limitation embraces practices such as the castration of male lambs and the isolation of rams and ewes except when mating is desired, and requires no further discussion here. Control of the time of mating and increasing the number of progeny are the main areas of interest in Australia. Induction of abortion and of parturition are seldom employed in Australia, but veterinarians are occasionally asked to perform one or other procedure.

Control of the time of mating

The basic requirements for an effective method of control of the times of oestrus and mating are:

Cyclic ewes

In these ewes control is achieved by either (a) artificially extending the ’luteal phase’ of the oestrous cycle by administering progesterone or other progestagens, or (b) prematurely inducing luteolysis, by giving prostaglandins. Figure 8.1 depicts the levels in peripheral plasma in the ewe of LH, oestradiol and progesterone during the normal oestrous cycle.

Figure 8.1 Levels of LH, oestradiol and progesterone in peripheral plasma of the ewe during the oestrus cycle.

The oestradiol level peaks at or just before the onset of oestrus and the LH peak occurs 6‑18 hours later. Oestrus lasts for about 20‑40 hours, being longer and more intense in multiple ovulating breeds. Ovulation occurs at around the end of oestrus. Thus the interval between the onset of oestrus and ovulation in Merinos is usually about one day, with considerable variation; but in more fecund genotypes it may be considerably longer. This has practical implications, in deciding when to hand mate or more especially inseminate different types of sheep in relation to the onset of oestrus (the time of ovulation will usually not be known).

The luteal phase progesterone (Figure 8.1) has three important functions in relation to the next ovulation. This progesterone ensures that :-

The peaks in oestradiol level that occur during the luteal phase are not required for the maintenance of pregnancy, but they permit non‑pregnant ewes to return to oestrus and have another chance to conceive. This luteal phase oestradiol has this effect in non-pregnant ewes by priming the uterus to respond to ovarian oxytocin. This, in turn, results in uterine PGF₂_α release and luteolysis.

Systems for oestrus synchronization should enable the insemination (or natural mating) of groups of ewes on a pre‑determined date and preferably at a pre‑determined time. If the degree of synchrony is sufficiently good, the ewes can be inseminated at a fixed time, without detection of oestrus. As well as requiring satisfactory synchrony and fertility in ewes regardless of the stage of the oestrous cycle at which treatment commences, the treatment should also produce a mild degree of superovulation, resulting in about an extra 0.5 ovulations per ewe. These extra ovulations should boost both conception and twinning rates. In practice, a ’trade‑off’ is often observed between the precision in timing and fertility. For example, giving progestagens for longer than the recommended period may enhance precision at the expense of reduced fertility.

Progestagens to synchronize oestrus

The use of progestagens is the preferred method of oestrus synchronization in the ewe. When, as is usual, the stage of the oestrous cycle is not known, treatment should be spread over at least 12 days and preferably be given for 12‑13 days. If treatment commences on day 5 or later in the cycle (day 0 = oestrus) the life‑span of the existing corpus luteum is unaltered. If treatment commences on day 4 or earlier, the time of luteolysis is advanced (to around day 12). Can you recall the physiological explanation of this phenomenon? In the past progestagens (including progesterone) have been given orally, by daily injection in oil or by a subcutaneous implant. However, these methods of administration generally give unsatisfactory results, with inadequate precision in the interval between cessation of treatment and oestrus (ie they do not give a sharp ’end‑point’ to treatment). The following two procedures have proved equally and highly successful and are commonly employed. Both can be considered to be portable, intra‑vaginal ’corpora lutea’ which can be inserted and removed at will:

Table 8.1 shows typical data for the interval between progestagen sponge removal and oestrus in cyclic ewes. Chronogest 30 mg sponges were used. PMSG was given to some ewes at the time of sponge removal to further stimulate folliculogenesis.

Table 8.1 : Interval between sponge removal and oestrus

The dose of PMSG depends mainly on bodyweight and breed (eg Merinos, 300‑400 IU; Border Leicester, 600 IU). The optimal dose of PMSG is determined only after some trial and error. These data are based on twice daily inspections for oestrus. The small dose of PMSG both advances and increases the precision in the time of onset of oestrus. It also slightly increases ovulation rate. The degree of precision obtained with PMSG should probably be adequate to allow insemination at a fixed time (around 48 hours) after sponge removal. Fertility at a controlled oestrus is usually reduced (as compared to a spontaneous oestrus), but returns to normal at the second, spontaneous and partially synchronized oestrus after treatment. The extent of the depression of fertility at the controlled oestrus is highly variable and results primarily from faulty sperm transport through the cervix. This defect can usually be overcome by depositing more semen in the ewe (use more rams for natural mating or more spermatozoa for cervical AI) or by depositing semen directly into the uterus (laparoscopic AI).

Prostaglandins for synchronizing oestrus

A single injection of a prostaglandin is likely to be effective in only about 70‑75% of ewes (those at Day 5 or later in the cycle). Possible alternative approaches to using prostaglandins are:

Cloprostenol, 100 μg, (Estrumate, Jurox Pty Ltd) or dinoprost‑PGF₂_α, 4‑5 mg, (Lutalyse, Upjohn Pty Ltd) are effective. Oestrus occurs 2‑3 days after treatment but not with sufficient precision to allow fixed‑time inseminations. Further, these treatments will be quite unreliable in any situation where a significant portion of the ewes are not cycling regularly. In consequence of these factors and in contrast to the case in cattle, prostaglandins are little used for controlled breeding in sheep.

Anovular (or anoestrous ewes)

In these sheep it is necessary to induce as well as control the times of oestrus and ovulation. Two distinct types of anoestrus occur (viz seasonal and post‑partum). In the context of the Australian sheep industry seasonal anoestrus is much the more important of these two states; many AI and MOET programmes, especially on studs, are performed outside of the breeding season. Post‑partum anoestrus becomes an issue only when lambing occurs at intervals of less than one year. This is quite common in some European countries, but unusual in Australia. Note that seasonal anoestrus may involve prepubertal ewes which would have reached puberty had seasonal anoestrus not intervened. Since Merinos and Dorsets are less rigorously seasonal than most other sheep breeds in Australia, it is easier to manipulate reproduction in these breeds and their crosses in the non‑breeding season.

There are basically four approaches that can be used to induce oestrus and ovulation in ewes in seasonal anoestrus. These are:

Various combinations of these four approaches are also possible. While approaches 2, 3 and 4 may lead to good conception rates and flock fertility after an out‑of‑season joining, responses are quite variable and are influenced particularly by breed and type, body weight/condition score, environment and month of joining. If natural mating is employed, more rams are required, especially in the case of British breed rams like the Border Leicester, Suffolk and Romney.

Melatonin implants to stimulate oestrus

Direct regulation of the light‑dark cycle is too costly and time consuming to find commercial use. The use of exogenous melatonin is much more practicable. The implants (Regulin, Regulin Ltd) are inserted subcutaneously near the base of the ear. Regulin is recommended for October, November and December joinings. The resulting increased plasma melatonin levels appear to simulate those which occur spontaneously after the summer solstice. The ewes are isolated from rams for 6 weeks before joining and receive a single implant 30‑40 days before joining for 6‑8 weeks. Hence these recommendations are utilizing both the light‑dark cycle and ram effects (ie Regulin seems to prime the ewes or reduce the depth of anoestrus, to respond better to the introduction of rams). Extensive trials have yielded the average results shown in Table 8.2.

Table 8.2 : Responses to Regulin

	per 100 ewes joined
	Merinos / Comebacks / Polwarths / Corriedales		First cross (BL/Mo)
	Untreated	Regulin	Untreated	Regulin
empty ewes	22	12	9	7
single bearing ewes	71	68	72	57
twin bearing ewes	7	20	19	36
triplet bearing ewes	0	0	0	0
lambs born	85	108	110	130
Period (days) in which 80% of ewes lambed	28	19	41	28

Note the considerable stimulation of ovulation rate. These data do not reveal substantial variation in the Regulin effect in individual flocks. The decrease in percentage of dry ewes induced by Regulin is more marked in maiden than in mature ewes. There may be little effect of Regulin in occasional situations where some Merino types show spontaneous oestrous cycles in late spring/early summer. Regulin gives poor results and is not recommended for use in the very seasonal British breeds. Regulin should not be used if the spring joining may have to be abandoned; such treatment appears to lower performance if the ewes are re‑joined in autumn.

Gonadotrophic hormones to stimulate oestrus

When suitably administered, gonadotrophins with FSH activity and GnRH (LHRH) will induce ovulation in the anoestrous ewe, but the ovulation is not usually accompanied by oestrus. If treatment is preceded by giving a progestagen for a minimum of 6 days, the ovulation is accompanied by oestrus. In practice the usual regime is to insert sponges or CIDRs for 12 days and give PMSG at the time of device removal. A slightly higher dose of PMSG than is used in the cyclic ewe is required. Sometimes the CIDRs are used for only 6 days, washed and re‑used! The use of progestagens plus PMSG is expensive. The response to such hormone treatment is critically influenced by stage of anoestrus (or month of joining). Table 8.3 lists some effects in (Border Leicester x Merino) XB ewes of stage of anoestrus, in terms of (a) % of ewes coming into oestrus (b) % of ewes in oestrus which conceive and (c) the probability of further breeding (or returns to oestrus) in ewes which do not conceive at the induced oestrus. The XB ewes were treated with a sponge or CIDR for 12 days and received 500‑700 IU PMSG at device removal.

Table 8.3 : Response of BL/Mo XB ewes to progestagens plus PMSG varies with stage of anoestrus

Stage of anoestrus	Effectiveness of treatment
	Ewes in oestrus (a)	Ewes pregnant (b)	Further breeding (c)
deep (Aug-Oct)	50% or less	50% or less	nil
late (Nov-Dec)	50-80%	50-60%	possible
very late (Jan-Feb)	80% or more	60% or more	probable

For natural mating, the number of rams required for large flocks may be prohibitive. The number can be much reduced by dividing the ewe flock into subgroups and staggering the hormone treatments by intervals of 2‑3 days. For best results, the number of ewes in oestrus per day per ram should not exceed three where British breed rams are used.

Figure 8.2 Seasonal pattern in the mean time to onset of oestrus following CIDR or sponge removal and treatment with 400 IU PMSG.

Equivalent tables for the effects of stage of anoestrus in Merino and British breed flocks are not shown. How would you expect the data in such tables to differ from those above for XB ewes? For Merinos, you would expect the same trend, but generally better results. For Nov‑Dec joinings, for example, 80‑90% should come into oestrus and perhaps 70% of these will conceive to the treatment; and further breeding is probable in animals which do not conceive at the induced oestrus. Conversely, in breeds such as the Border Leicester and Romney, the results will be worse, and there is little point in attempting controlled breeding until January‑February (very late anoestrus).

Returning to Merino and XB ewes, Figure 8.2 shows the influence of season on the mean time interval to onset of oestrus following sponge or CIDR removal and a fixed dose (400 IU) of PMSG. The interval is extended out of season, to about 40‑56 hours. The standard deviation for interval is also higher out of season. Of course, the interval is also sensitive to PMSG dose and, as this figure shows, type of progestagen device. Oestrus occurs about 10 hours earlier after CIDR than after the sponge. This probably reflects higher rates of metabolism and/or clearance of progesterone versus synthetic progestagens. The reduced precision obtained in times to oestrus and ovulation in ewes in anoestrus is an important reason why large scale AI programmes are best scheduled in or just before the normal breeding season (in very late anoestrus).

The ’ram effect’

The unaccustomed presence of rams with ewes can advance the onset of breeding activity, synchronize oestrus in groups of ewes to some degree, and may increase ovulation rate. Much out‑of‑season breeding of Merinos relies to a considerable extent on this ’ram effect’, although many producers are probably unaware of this mechanism. The ewes must be isolated from the sight and smell of rams for at least one month before joining. In Merinos, ram introduction in spring and summer (Oct‑Jan) causes a high proportion of ewes to breed. In the absence of conception, most will show 1‑3 consecutive cycles, then revert to spasmodic breeding or full anoestrus. When the ram effect works well, 80% or more of Merino ewes should lamb.

In breeds with a more sharply defined breeding season and deeper anoestrus, the rams must be introduced within 6 weeks of the time at which breeding would normally commence. If intro-duced earlier, the ewes’ responses are highly variable and usually poor. Sometimes the rams induce only an LH response, but not ovulation. There are big differences between rams in their ability to induce ovulation in anoestrous ewes. Merino rams are generally very potent. Experience in New Zealand has shown that Romney rams are fairly useless and Dorsets much better. Hogget rams are not as good as mature rams. The ram effect relies principally on pheromones secreted by the glands of the wool follicles. Their secretion is androgen dependent. These volatile chemicals (plus, perhaps, the sight and sound of rams?) increase the frequency of LH pulses in the ewe. This usually leads (if anoestrus is not too deep) to ovarian follicular development, oestrogen secretion and an LH surge, culminating in ovulation within 3 days of joining.

Figure 8.3 Ovarian responses in anoestrus Merino ewes to the introduction of rams. Varying portions of the ewe flock will exhibit Type I or Type II responses.

Merino ewes usually react to the introduction of rams in one of two ways. After the silent, ram‑induced ovulation on days 2‑3, (rams introduced day 0), most ewes experience a short cycle and have another silent ovulation on days 7‑8 (Type 1 response, Figure 8.3). This ovulation is silent because it is not preceded by an adequate period of progesterone priming. These ewes next experience a normal cycle, so that they ovulate a third time around days 24‑25, and this ovulation is accompanied by oestrus. The balance of the ewes in the flock do not experience the short cycle, so that after the ram‑induced ovulation they ovulate a second time around days 20‑21 and this ovulation is accompanied by oestrus (Type 2 response, Figure 8.3). The proportion of the flock exhibiting the short cycle is variable. In practice, there should be a high incidence of oestrus around days 19‑25.

For AI, testosterone‑treated wethers are best used. Even with natural mating, it is best, if feasible, to induce the ram effect with ’teaser’ wethers for the first 14‑16 days, so that Merino rams do not take out their frustrations on each other waiting for the ewes to come into oestrus. Testosterone cypionate or testosterone enanthate (Synarot, Banrot or Tesgro, 75 mg/ml, registered for the control in wethers of pizzle rot) is administered subcutaneously next to the base of the ear. Well grown, large wethers should be used. Note that, in this case, the wethers are only required to exude pheromones and not to mate actively with the ewes (contrast to the preparation of teasers for AI). A single dose of 400 mg given 14 days before use and 1% wethers are probably adequate. However, some veterinarians recommend using 2% wethers which have received 150 mg, 150 mg and 300 mg at 0, 7 and 14 days (inserting the ’teasers’ at 14 days and the rams at 28‑30 days).

The ram effect with ’social facilitation’

In New Zealand, where common ram breeds often fail to induce a strong ’ram effect’, there has been considerable interest in the ’social facilitation effect’. In this case a minor portion (eg 1/3rd) of the flock is treated with CIDR/PMSG so that they come into oestrus at the time of ram introduction. This strengthens the ram’s effect on the balance of the flock. The effect of the ewes in oestrus almost certainly acts via pheromones to stimulate the rams. The same effect may be seen when the rams are exposed to other ewes in oestrus prior to introducing the rams into the flock. This seems to be another expression of the quantitative nature of the ram effect. This is potentially a way to reduce the costs of hormone treatments for out‑of‑season breeding in New Zealand. There seems to be little interest in the ’social facilitation effect’ in Merinos in Australia.

The ram effect with progesterone pre-treatment

Table 8.4 shows two ways that the ram effect can be further exploited by certain hormonal pre-treatments of the ewes.

The use of a single dose of progesterone should cause essentially all ewes to come into oestrus around days 19‑20 (or ovulate on days 20‑21). This enables a relatively cheap approach to AI. Potentially, the whole flock may be inseminated over 4‑5 days, using teasers for oestrus detection. Fertility should be high, since it is a natural oestrus. However, depending on breed/bloodline and stage of anoestrus, only a variable proportion of the ewes may respond. The first or ram‑induced ovulation usually has a higher ovulation rate (an extra 0.2‑0.3 ova). Hence pre-treatment with sponges/CIDR not only advances the time of first oestrus by some 3 weeks, but also enables a higher twinning rate.

Table 8.4 : Effect of progesterone pre-treatment on ram effect and oestrus

Pre-treatment	Days after ram introduction
	2-3	7-8	20-21		24-25
NIL	OV	OV?	OV	or	OV
Progesterone; single 20mg dose at or 1-2 days prior to ram introduction	OV no OE	—	OV OE		—
Progestagen sponge or CIDR for 6-12 days, removed at ram introduction	OV OE	—	OV OE (returns)		—

In all cases the times of oestrus and ovulation are to a varying degree synchronized. In order to obtain high conception rates use 5% or more rams or stagger the ewes so that 3 of the ewes are introduced each 3‑4 days and use 2% rams. Prepubertal ewes which are of sufficient age and weight to have reached puberty will show the ram effect. What will be the resulting pattern of lambing? How will this be modified if a significant portion of Merino ewes are spontaneously cyclic at the time of ram introduction?

Oestrus stimulation in ewes in post-partum or lactational anoestrus

In ewes which lamb in the latter half of the breeding season, or in spring‑early summer, post‑partum anoestrus will usually extend into seasonal anoestrus. If ewes lamb just before or earlier in the breeding season, the first, silent post‑partum ovulation occurs 20‑30 days later. This first ovulation is usually followed by a short cycle and a second silent ovulation 6‑8 days later. This, in turn, is followed by a normal cycle and a third ovulation with oestrus at around 45‑55 days post‑partum. Note the similarity of this sequence of events in the post‑partum ewe to that seen in dry, seasonally anoestrous ewes after the introduction of rams. Hence, during most of the year, treatment to induce oestrus and ovulation will be necessary in order to breed recently lambed ewes. The recovery of the uterus post‑partum to provide an environment which allows fertilization and embryo development (involving a regrowth of an epithelial cell layer back over the caruncles) takes longer in ewes which are lactating and/or lamb in spring. More generally, the conception rate to a hormone‑induced oestrus and ovulation in post‑partum ewes is dependent upon:

In the irrigation districts of south‑eastern Australia attempts are occasionally made to produce prime lambs from XB ewes at intervals of less than one year. To be successful, such programmes require high standards of nutrition and management. Table 8.5 gives a guide to the pattern of return to normal fertility in BL/Mo XB ewes. It is important in such programmes that a high percentage of fertile rams are used (or, in the case of AI, ample doses of high quality semen). In practice, the minimal lambing interval achievable is about 200 days (3 lambings in two years or possibly 5 lambings in 3 years).

Table 8.5 : Days post-lambing for return of normal fertility

Increasing the number of progeny

The procedures available for increasing the number of progeny from rams and ewes are listed below and, in some cases, discussed. Some of the procedures have already been discussed; while AI, MOET and related techniques will be examined in detail later in this chapter.

The ram

The ewe

Before discussing some of these procedures in the ewe, it is well to repeat the general caution that the achievement of high fecundity in ewes (especially Merinos) requires higher standards of management and feeding and the acceptance of higher lamb losses.

Join at an earlier age

Joining at an earlier age requires a consideration of the factors which determine the age of puberty in ewes. These can be listed as follows:

Suppose, for example, that the breeding season in a highly seasonal breed is March‑June and the earliest age at which puberty is possible in well fed lambs in 8 months. In this case, the ewe lambs from ewes which conceive in March‑May could reach puberty at 8 months of age, but those from ewes which do not conceive until June cannot reach puberty until they are 16 months old. If we say that 10 months is the earliest age at which ewe lambs can reach puberty (which better approximates most situations in Australia) then the daughters of ewes which conceive in March can reach puberty at 10 months of age, but those of ewes which conceive in April‑June cannot reach puberty until 16‑18 months of age.

Select for increased litter size

Selection for increased litter size within a breed is possible but difficult and not often pursued in practice. Estimates of the heritability (h²) of number of lambs born vary but are usually about 0.15 and genetic progress in reproductive rate of up to 1.5% annually has been achieved by selecting rams on the dams’ reproductive performance. Genetic responses occur principally in ovulation rate. The increased OR seems to result from both (a) increased production of gonadotrophins and (b) increased ovarian sensitivity to gonadotrophins.

Cross-breed with more fecund types or breeds

Cross-breeding with more fecund breeds or types will probably also exploit heterosis. Much of the increase in lambing performance of (Border Leicester x Merino) XB ewes is due to heterosis, since lambing performance drops when the first‑cross is consolidated by interbreeding. The first‑cross produces 0.3 ‑ 0.5 more lambs per ewe joined than the mean of the two parent breeds. Crossing of Booroolas[1] with other Merino bloodlines increases ovulation rate and numbers of lambs born (especially if the Booroolas are homozygous for the Fec^B gene). Merino ewes heterozygous for the Fec^B gene typically have ovulation rates of 2.7‑3.1. However, depending on nutrition, the severity of the environment and perhaps predators, the number of lambs weaned often does not increase and may even decline. The distribution of OR in a Booroola cross‑bred flock will be complex, since the flock will contain FF, F+ and ++ ewes. The Fec^B gene has an autosomal locus. It has an additive effect on OR, but seems not to be expressed in the ram.

Induce a temporary increase in OR

The induction of a temporary increase in OR may be achieved by the use of progestagen sponges and PMSG, as previously discussed, aiming for a mean increase in OR of about 0.5. Alternatively, various immunization procedures can induce a short‑term increase in OR. For some years the vaccine Fecundin was available to sheep producers. The immunogen in Fecundin, polyandroalbumin, contained androstenedione as an antigenic determinant. Vaccination resulted in decreased plasma free androgen (and probably oestrogen) levels and an increase in gonadotrophin secretion. Vaccination was carried out 5 and 2 weeks before joining for 6‑8 weeks; in subsequent years a booster dose only was given to the flock 2 weeks before joining, depending on the season and the desirability of obtaining more twins.

The results achieved in terms of lambs weaned were somewhat erratic and the vaccine never became very popular, for several reasons. Commonly the subsequent management of flocks was not good enough to permit high survival rates in multiple‑status lambs. Ovarian responses were generally better in high rather than low condition score ewes and in XB rather than Merino ewes. It was generally wise to not treat maiden ewes. Vaccination did not correct a fertility problem and there was no decrease in the number of dry ewes. Research studies suggested that vaccination sometimes increased the rate of embryo mortality, possibly by altering the oviductal/uterine environment. Other research studies have shown that a modest increase in OR can be obtained in ewes by vaccination against inhibin, an ovarian regulator of FSH secretion. Perhaps a commercial vaccine based on inhibin or an inhibin agonist will become available in the future?

Induction of abortion

The maintenance of pregnancy in the ewe ceases to be dependent on the corpus luteum at around day 50. Prior to this time, abortion can be reliably effected with a single luteolytic dose of prostaglandin (for doses, see earlier). In cases of misalliance, the earlier treatment is given the better, but treatment before day 5 is ineffective. After day 50 abortion is not easily induced with even large and/or repeated doses of prostaglandins. These treatments with higher doses are dangerous and the retention of foetal membranes is a serious side‑effect. Glucocorticoids are unreliable before day 140. You should dissuade your client from attempting treatment after day 50. If another pregnancy is required, allow the ewe to go to term and then remove the lamb.

Induction of parturition

Parturition is usually induced with glucocorticoids. This should initiate most of the normal prepartum maturational events, thereby ensuring adequate foetal preparation for postnatal life. 15 mg dexamethasone should result in parturition some 24‑48 hours later. The optimal treatment time depends on breed. Treatments on days 144 and 148 are recommended for British breeds and Merinos, respectively. Following a synchronized mating, the treatments are given when 5% of the flock have lambed. Some increase in the rate of perinatal mortality should be expected after these treatments.

Artificial insemination (AI)

Only a very small proportion of ewes in Australia are artificially inseminated. It is difficult to get accurate figures on the use of AI. Nevertheless, it appears that some hundreds of thousands of ewes are artificially inseminated annually and that the majority of these inseminations are carried out or supervised by veterinarians. Increasing significance is being given to progeny testing of rams and the sale of semen by Merino studs. As an historical trend, the role of veterinarians has shifted away from large‑scale AI programmes in autumn using natural heat detection and cervical insemination and towards more compact or concentrated programmes using controlled oestrus and uterine insemination. These programmes, which are usually on studs, are often required in December ‑ February. It is also reasonably common in some districts for commercial wool producers to ask veterinarians to inseminate (uterine AI) 100 (say) ewes with purchased semen, to produce their own superior young rams. Allowing for 60% culling of ram lambs, this should yield about 10‑12 replacement rams at a cost that is often below that of buying in replacements. This exercise is probably only worthwhile where the flock size exceeds 2000. Successful involvement of veterinarians in sheep AI programmes requires good planning and organization and an uninterrupted allocation of time, which is often difficult in a busy practice situation.

Selection and preparation of rams

This requires much care. Rams should have a thorough clinical examination to check both general health and the reproductive organs (Chapter 7). A test for brucellosis may be necessary. Collect a semen sample by electroejaculation to confirm the presence of ample normal, motile spermatozoa. Assess any information about the genetic merit and commercial value of the ram. Allow sufficient time between selection and use for training to the artificial vagina, and check ram management, especially feeding and conditions of shedding. Be wary of the stud breeder who wants a salvage operation for a ram which is not performing well in natural mating.

Collection of semen

Two procedures only are potentially available. The relative advantages and disadvantages of using electroejaculation and the artificial vagina are as follows:

Electroejaculation

Artificial vagina

In summary, electroejaculation is the procedure employed on most sheep properties, where semen collection is required as an adjunct to ram soundness and fertility investigations, whereas the artificial vagina must be used for any significant AI programme. Electroejaculation is achieved by stimulating per rectum the nerves around the accessory glands and the base of the penis. The artificial vagina should simulate the characteristics of the ewe’s vagina, in respect of temperature, pressure and lubrication. The apparatus and procedures required for each method of collection, as well as the procedure for training rams to the artificial vagina, will be examined in the practical classes.

Handling of semen

A number of factors may adversely affect the viability of semen during handling. These are:

Change of temperature

A sudden drop in temperature to less than 10EC causes ’cold shock’; an irreversible loss of viability. Exposure to temperatures greater than 42EC causes rapid death of spermatozoa, whereas storage at greater than 30EC reduces the fertilizing life of the sperm, due to exhaustion of energy sources and a drop in pH (resulting from the accumulation of lactic acid). An ideal temperature for short term (1‑2 hours) storage is 25‑30EC. Slow cooling over 2‑3 hours to around 5EC will maintain good viability and fertilizing capacity for at least 12 hours.

Sunlight

Exposure to direct sunlight damages spermatozoa, mainly due to the effects of ultraviolet radiation.

Exposure to heavy metals

Copper, lead, mercury and cadmium are toxic to spermatozoa; glass or plastic containers should be used for semen.

Contact with water

The rapid change in osmotic pressure kills spermatozoa. Artificial vaginas and collection and storage vessels must be dry.

Contaminants

Disinfectants

Nearly all antiseptics, bactericides and detergents are spermicidal. What are the preferred cleaning and sterilization procedures? Glass or plastic containers should be washed thoroughly, then rinsed repeatedly and thoroughly with distilled water to remove detergents. Sterilization by dry heat, gas (ethylene oxide) or UV irradiation is effective. There is a danger with autoclaving of contamination with heavy metals or other materials previously placed in the autoclave.

Evaluation of semen

Semen can be directly evaluated by measuring pregnancy or lambing rates but in practice the substantial costs and time lags involved are major problems. Thus the indirect evaluation or assessment of semen in the field is a critical factor to the success of any AI programme and expert opinion should be sought by inexperienced veterinarians.

Table 8.6 : Scoring system based on macroscopic examination

Estimates of probable fertilizing capacity can be made by visual examination and, if necessary, various biochemical tests. Semen samples can be examined macroscopically for volume, colour, consistency (or density) and motility. Average ejaculates to the artificial vagina have a volume of around 1.0 ml (range 0.5 ‑ 1.5 ml). The colour should be milky white; other colours indicate contamination. The consistency of the ejaculate can be scored subjectively, according to the criteria in Table 8.6. Motility can be only roughly assessed macroscopically; swirling on the side of the collecting vessel indicates high motility.

Microscopic examination of semen may be used to determine motility, density, % dead sperm, morphology and viability. Motility can be scored subjectively, with the criteria in Table 8.7.

Table 8.7 : Scoring system based on microscopic examination

Score	Motility	Percent of sperm active	Description
5	excellent	>80	rapid, dense waves changing direction rapidly
4	good	65-80	good wave motion but slower changes in direction
3	fair	40-65	slow thin waves
2	poor	20-40	no waves; individual sperm can be seen
1	very poor	<20	few sperm show progressive movement
0	none	0	no movement

Scoring must be carried out on a warm slide (37EC), preferably on a warm stage and at low magnification (40‑100x) with a thin sample. Score rapidly, because the sample will soon dry. Assessment of wave motion is difficult or impossible where semen has been extensively diluted or frozen and thawed; in these cases the percentage of progressively motile sperm is estimated. In practice the subjective scoring of semen samples for density and motility is usually sufficient in a commercial AI programme. Microscopic or other objective determination of density (haemocytometer, colorimeter, packed cell volume) and determination of % dead sperm (stain method), morphology and viability after in vitro incubation and biochemical tests (respiration, fructolysis, pH, et cetera.) are not usually necessary, and will not be detailed here. Every semen sample contains a proportion of abnormal sperm. In good quality ejaculates the percentage may be 5‑10%. Increasingly, the use and sale of ram semen is being underpinned by objective measurement of semen characteristics. Commercial laboratories use computerized image analysis systems to give accurate information on submitted semen samples. This enables measurement of progressive motility and velocity, motility, motile sperm concentration, et cetera. Progressive motility is the proportion of spermatozoa moving forwards at more than a defined speed. Some healthy caution is still required is assessing the practical significance of these various objectively measured semen characteristics. What significant correlations have been demonstrated between objectively measured characteristics and fertility? There is some experimental evidence for significant correlations, but a reliable in vitro test for prediction of fertility still eludes us.

Dilution of ram semen

The dilution of ram semen is generally essential but may not be necessary if only a modest number of ewes are to be inseminated promptly after semen collection. Semen is diluted for both technical and biological reasons. The technical reason is to enable more ewes to be inseminated with a minimal volume of inseminate (in effect, to get the right number of motile sperm into the right volume for insemination). The biological reason is to enhance the survival of sperm, by:

Formulae for the preparation of various diluents suitable for both the fresh use and freezing of ram semen can be found in the text by Evans and Maxwell (1987). Antibiotics should be added to all diluents (eg penicillin, 200 IU/ml; streptomycin, 1.0 mg/ml). For the fresh use of ram semen, various synthetic diluents, including Dulbecco’s phosphate buffered saline, or UHT cows’ milk can be used. Other ty pes of cows’ milk require pre‑treatment to inactive toxic factors. For the busy veterinarian, freshly opened UHT milk is probably the diluent of choice, for both cervical and uterine insemination. Note the following points about the dilution procedure:

Evans and Maxwell (1987) provide recipes for diluents containing glycerol and egg yolk which are suitable for one‑step freezing of ram semen in pellet form or in straws. The composition of these diluents may have to be adjusted for different dilution rates. All presently available freezing diluents seem to reduce the quality of frozen‑thawed spermatozoa and there is ongoing research to devise superior diluents.

Storage of ram semen

Semen must be stored in a manner which decreases or arrests metabolism and hence prolongs the fertilizing life of the sperm. Semen is stored either (a) chilled, at 2‑5EC, or (b) frozen in pellets or straws and held at ‑196EC. For liquid or chilled storage the semen is cooled slowly over 2‑3 hours. For use, it may be warmed promptly to 30EC or inseminated without warming. Chilled semen maintains good motility (as seen in re‑warmed samples) for several days. However, the fertilizing ability of this semen after cervical insemination declines progressively, so that for acceptable fertility (>45% conception rate?) the semen should be stored for not more than 12‑24 hours. If uterine insemination is used, the semen may be used after storage for up to 3 days. For frozen storage, the semen is diluted with diluent containing cryoprotectant and chilled to 2‑5EC, as before.

Two methods of freezing and thawing are used. In the pellet method, the rate of temperature change is fast. Aliquots (0.2‑0.5 ml) of chilled semen are dropped into holes made on the surface of a block of dry ice (solid CO₂) and the resulting pellets are transferred with forceps into liquid nitrogen. For use, the pellets are transferred promptly into a dry vessel at 37EC to obtain rapid thawing and maximum recovery. In the straw method, which is semi‑fast, the straws are loaded with chilled semen and placed in liquid N₂ vapour; then stored in liquid N₂. For use, the straws are thawed at 20‑30EC. The proportion of spermatozoa which survive these procedures (as indicated by subsequent motility) varies widely. 40‑70% should survive in good semen samples. Semen from some individual rams will not tolerate freezing and thawing. The inseminating dose must be adjusted according to the survival rate (12 ‑ 2x number of sperm inseminated fresh). The conception rates obtained after cervical AI are only 30‑40% at best and usually lower. Sperm which survive the procedure have a decreased fertilizing life. Uterine AI is always recommended, and conception rates by this route can be good. The procedures for freezing, storing and thawing semen are examined in more detail in the practical classes.

Detection of ewes in oestrus

An important decision in any AI programme is whether to inseminate to oestrus detection or at a fixed time after treatment. Even where the latter policy is adopted, it is probably worthwhile to run raddled teasers with the ewes and inseminate first those ewes which came into oestrus first, since the inseminations may take many hours or all day. In any event, you will probably want to run some teasers with the ewes to monitor the success of the hormone treatments in the ewes, and it is possible that the presence of teasers enhances the ewes’ responses. Where insemination is based on oestrus detection, the quality of detection will clearly be an important determinant of the conception rate to AI. Detection is necessarily based on the use of teasers with crayon bearing harnesses. The selection, preparation and management of teasers were previously discussed. The following percentages of teasers are recommended:

Rams are occasionally used as teasers. They may be entire rams fitted with aprons, but this system is not reliable. Alternatively, sexually active young rams are vasectomized at least 6 weeks before use, and immediately before use semen samples are collected by electroejaculation to confirm the absence of spermatozoa. Techniques employing caudal epididymectomy or penis sheath deviation are not recommended. The teasers used today are usually testosterone‑treated wethers. If the wethers are given 150 mg testosterone cypionate (or enanthate) at fortnightly intervals, commencing 4 weeks before use and continuing until the end of the AI programme, they should exhibit good mounting activity.

Insemination of the ewe

During coitus, semen is deposited in the vagina. Ejaculation is rapid, with a small volume of semen containing a high concentration of sperm. AI should at least simulate this and preferably deposit semen in a more favourable position for fertilization (cervix, uterus). However, in the ewe it is very difficult and usually impossible to pass an inseminating pipette through the cervix. Three methods of insemination are employed in Australia; vaginal, cervical and uterine. The apparatus and facilities required and the procedures involved for each of these three methods are examined in the practical classes.

The volume of inseminate can vary slightly within set limits. The upper limit will be determined by the capacity of the organ or site of insemination to retain the semen. Volumes below 50 μl are not practicable. Recommended volumes are:

The minimal safe numbers of motile spermatozoa in the inseminate (required to obtain satisfactory conception rates) are determined by the route of insemination, type of semen (fresh/liquid‑stored/frozen‑thawed) and whether oestrus in the ewes is spontaneous or controlled. Some workers have obtained good fertility with considerably lower numbers of sperm, but the numbers of motile sperm shown in Table 8.8 are recommended for general use.

Table 8.8 : Minimal safe numbers of motile spermatozoa per inseminate

Route of insemination	Type of oestrus	Type of semen
		Fresh (x10⁶)	Liquid-stored (x10⁶)	Frozen-thawed (x10⁶)
vaginal	spontaneous ontrolled	150 300	NR NR	NR NR
cervical	spontaneous ontrolled	100 200	150 300	NR NR
Uterine per uterine horn)	controlled superovulated	15 20	15 20	20 30

NR = not recommended; conception rates generally <50%, may be very low. If ample semen is available, the number of spermatozoa can sensibly be increased somewhat above the relevant number shown above.

The time of insemination is related to the time of ovulation. Ovulation occurs around the end of oestrus. Insemination should be performed at a time sufficiently before ovulation so that by the time of ovulation a large population of sperm is established in the ampulla, the site of fertilization. In general, this means insemination 12‑24 hours before ovulation. However, time of insemination is adjusted according to whether the semen is fresh or frozen‑thawed, since the former should have a fertilizing life in the female reproductive tract well in excess of 24 hours, while for the latter the fertilizing life may not be more than 12 hours. For insemination of ewes in spontaneous oestrus (vaginal or cervical AI), aim for 12‑18 hours after the onset of oestrus, with twice daily inspections for detection. With once daily inspection in the morning, ewes in oestrus should be inseminated soon after inspection.

For insemination of ewes in controlled oestrus (cervical or uterine AI), insemination is usually performed at a fixed time in relation to the synchronization treatment. (However, some commercial operators still prefer to inseminate in relation to onset of oestrus). The interval between progestagen device removal and time of insemination is influenced by season, type of intra‑vaginal device (shorter for CIDR than progestagen sponge), type of semen (fresh, frozen), the use of PMSG and the dose employed. If progestagen sponges are used in conjunction with a non‑superovulating dose of PMSG and 5‑10% teasers, the majority of ewes will be in oestrus within 36‑48 hours and ovulate about 60 hours after sponge withdrawal. Superovulated donor ewes for MOET programmes, which have been treated with larger doses of PMSG and/or other gonadotrophins will be in oestrus within 24‑36 hours and ovulate about 48 hours after sponge withdrawal. Insemination is recommended at the following times after progestagen sponge withdrawal, where a single insemination is employed:

cervical insemination	+ PMSG, fresh semen	36‑48 hours
" "	‑ PMSG, fresh semen	48‑60 hours
uterine insemination	+ PMSG
(not superovulated)	fresh semen	36‑48 hours
" "	frozen semen	60‑66 hours
uterine insemination
(superovulated)	fresh semen	24‑ 48 hours
" "	frozen semen	44‑ 54 hours

These times can all be shortened a little if CIDRs are used instead of progestagen sponges.

Ewes are usually inseminated only once. Double inseminations are not recommended where uterine insemination is used. In the case of cervical insemination, you must balance the likely small (5‑10%) increase in conception rate against the extra costs associated with double insemination. If employed in ewes not receiving PMSG, inseminate at 48‑50 and 58‑60 hours after sponge removal. Use the recommended number of motile sperm at each insemination.

What conception rates can reasonably be expected? Done carefully, they should be about the same as those that could be obtained with natural mating with ample ram power. But in practice they are commonly somewhat less. You will hear occasional claims from commercial operators of extraordinary conception rates (up to 90%), but treat these claims cautiously. Rates of 60‑70% using uterine AI are very acceptable and seem to be often achieved by better operators.

Overall planning and management of an AI programme

Individual AI programmes may involve as few as 5 or as many as 20,000 ewes. The larger programmes are laborious and expensive, but also very challenging. They represent a major investment on the part of your client. In the case of uterine AI the equipment that you require is expensive and some time is needed to develop expertise. Veterinarians will probably need to do at least 1000 animals per annum to maintain their skill and justify this investment. However, do not be too ambitious initially, as small ’crashes’ are better than large ’crashes’! The optical telescope in particular is delicate and liable to be damaged, so a spare should always be carried. In contrast, vaginal insemination programmes, which are still used to a limited extent, require little equipment and can be carried out by one person. There seems to be little role for veterinarians in these programmes.

A full discussion of planning and management is beyond the scope of this chapter. However, some of the issues that need to be addressed are indicated by the following questions:

It should be clear that the programme does not end on the day or days of insemination. In view of the high costs involved ’up front’, extra attention must be given to subsequent flock management in order to maximize lamb survival. Remember that because of the treatments employed, the twinning rate is likely to be higher than is normal for the type of sheep involved, and most of the lambs will be born over just a few days.

Multiple ovulation and embryo transfer (MOET)

The ovaries of the ewe contain vast numbers of oocytes, only a minute proportion of which ever develop into lambs. Almost all the oocytes become atretic and are lost. This represents a loss of potentially valuable progeny in genetically superior ewes. MOET provides a method whereby some of these otherwise wasted oocytes can be used. The successful planning and execution of MOET programmes in sheep are difficult and challenging work for veterinarians. The level of usage of MOET in sheep in Australia remains modest, for several reasons, including:

Clients are usually poorly informed of the underlying biology, the procedures involved and the potential benefits and costs. These issues should be discussed carefully with any enquiring client. You should consider how many donors the client can afford to submit to a programme, and how many donors you or your team can sensibly handle. Depending on the resources available, the embryos from 5‑10 donors can be comfortably collected and transferred in one day. To programme less than 5 donor ewes risks too much variation in embryo yield. The standard of general husbandry should be excellent. Donors and recipients should be shorn and managed to be in condition score 22 - 32 and on above-maintenance feed. For the surgical procedures, clean up the shearing board area thoroughly and ensure that good lighting is available. The apparatus and facilities required and the procedures involved in MOET are examined in the practical classes. The technique involves the collection of early embryos from superovulated donor ewes and their assessment and transfer to synchronized, recipient ewes.

Superovulation of donor ewes

Various gonadotrophins rich in follicle stimulating activity will induce superovulation when given during the latter stages of the luteal phase of the oestrous cycle. PMSG used to be the gonadotrophin most widely used in practice, because of its availability, relative cheapness and ease of use. However, when used alone at doses of 1200‑1500 IU, a significant proportion of ewes show ovarian responses characterized by many persistent large follicles which fail to ovulate and relatively few ovulations. Hence the dose response line for ovulations is not linear. Supplementation of PMSG treatment with GnRH or antiserum or monoclonal antibody to PMSG can reduce the incidence of unovulated follicles, but these treatments are rarely used commercially.

Purified FSH of porcine, bovine or ovine origin is also available commercially and is widely used for superovulation. It gives more ovulations and fewer persistent large follicles than PMSG, but its use is more tedious and some ewes fail to exhibit any superovulatory response (regardless of the dose of FSH employed). FSH has a half‑life in the sheep of around 2 hours, which is much less than the equivalent interval of about 20 hours for PMSG. PMSG is given by a single intramuscular or subcutaneous injection, whereas FSH, because of its much shorter half‑life, has to be given as a series of injections, usually twice daily over 3 consecutive days.

A ’cocktail’ of FSH and PMSG seems now to be the preferred superovulatory treatment. In this case, both hormones can be given as single injections at the same time (eg 500‑800 IU PMSG plus 12‑20 mg FSH). There seems to be a higher ovulation rate and more transferable embryos than when either FSH or PMSG is used alone at equivalent doses, and, compared to using FSH alone, there is an increase in the percentage of ewes exhibiting a superovulatory response. Some trial and error may be necessary to determine the optimal doses of PMSG and FSH in the ’cocktail’.

In order to effectively programme the transfer operations, it is essential to control the times of oestrus and ovulation in donor ewes, preferably by using progestagen sponges or CIDR for 12‑13 days. If PMSG alone or a ’cocktail’ of FSH and PMSG is used, it is given 36‑48 hours before removal of the intravaginal device. The ewes should be in oestrus 24‑36 hours after sponge removal, and, at least following PMSG or ’cocktail’ treatment, the ewes probably ovulate around 42‑54 hours after sponge removal. However, several factors appear to influence the timing and spread of ovulations following a superovulatory treatment, and some experience is required in determining the optimal time for insemination. The uncertainty about the time of ovulation is a real problem where frozen semen is employed. There is a good case here for a supplementary GnRH treatment, to reduce the interval from first to last ovulation and to shorten the time elapsing between sponge removal and the median time of ovulation. For example, 20‑40 IU GnRH (eg Fertagyl, Intervet) are often given at 24 hours after sponge removal. There is probably no need to use GnRH where fresh semen is employed. Suggested optimal times for insemination were listed previously. In the case of fresh semen, inseminations at 24 hours after sponge removal appear to give higher ova recovery rates but slightly lower fertilization rates than do inseminations at 48 hours. Can you suggest why this may be so?

Variation in the superovulatory response

As already noted, even today the variability in ovarian responses (numbers of ovulations and persistent large follicles which fail to ovulate) remains substantial and it is normal for some treated donors to yield no transferable embryos. It is important to realise that in the practice of MOET it is relatively easy for you to master the surgical and manipulative skills required and considerably more difficult to gain precise control over the superovulatory responses. Factors which influence the ovulatory response in donors include:

Mating

Fertilization in donors following natural mating or cervical insemination can fail, due to faulty transport of sperm through the cervix, following exposure of the cervix to abnormally high levels of ovarian hormones, principally of oestrogens. The problem is overcome by uterine insemination. It is a good idea to run raddled teasers with the donor ewes, for reasons that were previously outlined.

Embryos

The surgical collection, recovery from the flushing medium, assessment and storage of embryos are examined in the practical classes. Embryos are usually collected 3‑7 days after mating. Collections may be done by either (a) backward flushing of the uterine horns and oviducts or (b) flushing from the tip of the uterine horn only to a suitable catheter inserted at the external bifurcation of the horns. The latter approach is less invasive and can be used repeatedly in the same ewe, but it yields lower recovery rates and can be used only after day 4. Balanced salt solutions (eg Dulbecco’s phosphate buffered saline, pH 7.3) supplemented with 10% sheep serum and antibiotics are commonly used for flushing and storage. Veterinarians usually use complete, commercially prepared media. Fresh medium is used each day and stored at 30EC for use. Embryos should be located in the flushing medium and transferred to fresh medium early after flushing. They can then be stored in the medium at 20‑25EC for 3‑4 hours. A stereoscopic microscope (magnification 40‑60x) is used to locate and assess the embryos in the flushings and fresh medium.

The assessment of embryo viability is done morphologically and is based on general appearance and stage of development. This method is quick, cheap and non‑invasive, but subjective and the correlation with viability is certainly less than perfect. In the evaluation, consider size, fragmentation and granulation of blastomeres, symmetry of the cell mass and the appearance of the zona pellucida. There is no substitute for a lot of practice and determination of subsequent lambing rates. Systems for scoring embryos for quality or viability are often used, but are probably of limited value for the inexperienced.

Variation in the numbers of normal embryos recovered from donors

The recoveries from individual donors can range from none to 20 or more. Factors responsible for the substantial variation observed include:

Transfers to recipients

Transfers to recipients should be carried out as soon as reasonably possible after collection. The recipients must be in oestrus within 12 hours of their respective donors. Note that this implies that synchronizing intravaginal devices must be removed from recipients about 24 hours before they are removed from donors. Recipients need not be of the same breed as the donors. Mature (BL x Mo) XB ewes make attractive recipients, especially if two embryos are transferred to each recipient. Transfers are best done using a cradle, local anaesthesia and laparoscopy. A uterine horn ipsilateral to an ovary containing a corpus luteum is identified and then about 5 cm of the ovarian end of this horn is exteriorized through the wound made by the trochar for the transfer. There is always debate about the relative merits of transferring one or two good quality embryos per recipient. It seems reasonable to assume that pregnancy rates are higher after the transfer of two embryos, but in this case there may be a lower rate of conversion of embryos into live lambs. Thorough studies of this question are lacking. In commercial practice, decisions are often made ’on the run’ and are based on the relative numbers of embryos and recipients available, and the viability scores allotted to embryos. For maximal speed and efficiency in commercial MOET programmes it is best to deposit two embryos per recipient. Some operators using this policy claim 60% survival of transferred embryos. Programme five recipients per donor (this allows effective use of up to 10 embryos per donor). The general approach with retarded embryos is to transfer these last if recipients are available.

It should be self evident that, even more so than in the case of an AI programme, the subsequent feeding and management of the recipients during pregnancy, lambing and lactation should be excellent, in order to optimize lamb birth weights and maximize lamb survival.

Expected results for a MOET programme

The following results should be attainable in a well managed programme, but in practice the average number of progeny per treated donor seems often to be less than 4‑6.

* Assumes transfer of one or two normal embryos freshly collected from a mature donor; the survival rate will be lower after transfer:

(d) where the recipient was detected in oestrus more than 12 hours apart from the respective donor.

** This number can be increased to, say, 20 progeny per donor per annum, if the same donor is re‑programmed 4 times in the same year.

Schedule for the preparation of donor and recipient ewes

At this stage it should be useful revision to construct a calendar which shows the days and times of all treatments and procedures carried out on the donors and recipients in a MOET programme. The finer details of such a calendar will obviously vary, for reasons which have been discussed. Table 8.9 shows a sample calendar.

Storage of embryos

Sheep embryos can be successfully stored frozen in liquid nitrogen, probably for unlimited periods. At best, with present methods, about 70% of embryos survive frozen storage. Hence, in the case of frozen embryos, you could expect about 40‑50% to survive to lambs after transfer to synchronized recipients. The techniques used for freezing and thawing are still evolving. Slow and rapid procedures, as well as vitrification, are employed. There is only very limited commercial use of these procedures in Australia, mostly associated with the international transport of sheep embryos under quarantine conditions. Interest is inhibited by the low survival rates and restrictions on the export of Australian Merino embryos.

Table 8.9 : A sample calendar for a MOET programme

Day of programme	Donor ewes	Recipient ewes
0 (Thurs)	insert progestagen sponge or CIDR	insert progestagen sponge or CIDR
11 (Mon)	treat with 1200 IU PMSG or a ’cocktail’ of FSH & PMSG	—
12 (Tues, am)	—	remove sponge and treat with 400 IU PMSG; join harnessed teasers
3 (Wed, am)	remove sponge, join harnessed teasers	—
13 (Wed, pm)	isolate from feed and water	—
14 (Thurs, am)	optional GnRH treatment	—
14 (Thurs, am/pm)	oestrus	oestrus
14 (Thurs, pm)	intra‑uterine insemination with fresh semen	—
15 (Fri)	intra‑uterine insemination with frozen‑stored semen, remove teasers	remove teasers
19 (Tues, pm)	isolate from feed and water	isolate from feed and water
20 (Wed)	collect and evaluate Day 6 embryos	transfer one (or two?) normal embryo to a uterine horn ipsilateral to an ovary containing a corpus luteum
26-28	treat with luteolytic dose of prostaglandin analogue; remove skin sutures	—
67-69	—	score for pregnancy and twins using real‑time ultrasound (equals days 53‑55,if pregnant)
160	—	pregnant ewes will all lamb over the next 1‑7 days (if lambs are Merinos); review management of lambing

Some other technologies in sheep related to AI and MOET

These technologies generally remain somewhat experimental and so far have found little or no commercial application. Nevertheless, some promise exciting developments in sheep breeding. The technologies include the following.

In vitro culture of embryos

Success depends on the medium used, stage of embryo at commencement of culture and duration of culture. For example, the viability of day 5 embryos cultured in vitro from the zygote stage is now quite good for some flocks, but is always poorer than for control embryos cultured in vivo. How well do available media and incubation conditions simulate in vivo conditions? Co‑culture with oviductal or uterine epithelial cells can improve the results obtained. Embryos can also be cultured in vivo, in ligated rabbit or sheep oviducts.

In vitro maturation and fertilization of oocytes (IVM and IVF)

This requires optimal in vitro conditions for the maturation of follicular oocytes, capacitation of spermatozoa and fertilization. There is an enormous potential for IVF of oocytes collected from abattoir material. There has been much recent progress in developing these methodologies.

Insertion of desirable transgenes

Genes regulating fecundity, wool growth and other production traits are of interest. The insertions are usually made into zygotes at the pronuclear stage.

Embryo splitting

Embryo splitting is best carried out on late morulae and blastocysts (days 6‑10). The split late morulae or blastocysts should develop directly in final recipients without a zona pellucida. This procedure potentially enables an increase in the numbers of offspring in an MOET programme. The embryos are split with a fine glass needle or blade. There is a strict limit to how many times splitting can be repeated. The embryonic anti‑luteolytic signal in recipients will be diluted, so it is wise to transfer in pairs to increase the strength of the signal. If embryos are split into portions of less than 3 embryo, trophoblastic vesicles (lacking an inner cell mass) rather than normal embryos are likely to develop.

Nuclear transfer and clone formation

Morulae (16‑32 cells) are used as nuclei donors for cloning. Alternatively, embryonic stem cells in suitable culture potentially represent an inexhaustible source of genetic material for nuclear transfer cloning. Clone formation requires good techniques for the maturation and enucleation of oocytes, the fusion of nuclei with anucleated oocytes and in vitro culture of the resulting embryos to the late morula/blastocyst stage. ’Electrofusion’ is used to activate the oocyte during nuclear transplantation. Culture of the resulting embryos in ligated sheep oviducts may be preferable to in vitro culture.

Semen sexing and ICSI

The possibility of separating X‑ and Y‑chromosome bearing spermatozoa has long tantalized animal science researchers. The production of sexed semen on a scale sufficient for normal AI still is remote, but the successful separation of relatively small numbers of spermatozoa that can be used for IVF has been achieved in a number of species.

At present, there is a very low yield of correctly sexed cells from the flow cytometric sorting process used to separate the X and Y sperm. Fertilisation of in vitro matured oocytes by injection of a single spermatozoon (intracytoplasmic sperm injection or ICSI) is a method for producing viable embryos with a very small number of sperm. This technique has been widely used in human assisted reproduction but only a small number of offspring has been produced by ICSI in sheep.

Embryo sexing

The available methods include cytogenetic analysis, male‑specific (H‑Y) antigens and Y‑specific molecular probes. In practice, hybridization using Y‑specific DNA sequences and 8‑10 biopsied trophoblast cells seems to be the method of choice. There are significant problems of cost, loss of viability of embryos after biopsy, and inaccuracy. The scope for commercial application of embryo sexing in sheep is questionable. It will halve the efficiency of existing MOET procedures, assuming embryos of only one sex are required.

Juvenile In Vitro Embryo Technology (JIVET)

The production of offspring from very young pre-pubertal ewes has the potential to increase genetic gain by reducing the generation interval. Although still under experimental development, large numbers of viable embryos have been produced from superovulated lambs as young as 5 weeks of age. If commercially applied, JIVET has the potential to not only reduce generation intervals but also dramatically increase the efficiency of MOET programs in sheep and cattle.

General references

Cognie Y (1990) Current technologies for synchronization and artificial insemination of sheep; In Reproductive Physiology of Merino Sheep Eds CM Oldham GB Martin and IW Purvis, University of Western Australia, p 207

Evans G and Maxwell WMC (1987) Salamon’s Artificial Insemination of Sheep and Goats Butterworths, Sydney

Geldard H (1984) Field evaluation of Fecundin: an immunogen against androstenedione Proc Aust Soc Anim Prod 15 185

Holt NA (1988) Semen collection and storage AI programs and embryo transfer In Sheep Health and Production Proc 110 Post Graduate Committee in Veterinary Science, p 517

Hunter RHF (1980) Physiology and technology of Reproduction in Female Domestic Animals Academic Press, London

Killeen ID (1983) Artificial insemination and synchronization of oestrus In Sheep Production and Preventive Medicine Proc 67 Post Graduate Committee in Veterinary Science, p 302

Lindsay DR and Pearce DT (eds) (1984) Reproduction in Sheep Australian Academy of Science, Canberra

Maxwell WMC, Szell A, Hunton JR and Ryan JP (1990) Artificial breeding: embryo transfer and cloning In Reproductive Physiology of Merino Sheep Eds CM Oldham GB Martin and IW Purvis University of Western Australia, p 217

Miller SJ (1981) Artificial breeding techniques in sheep In Sheep Proc 58 Post Graduate Committee in Veterinary Science, p 71

Signoret JP (1990) The influence of the ram effect on the breeding activity of ewes and its underlying physiology In Reproductive Physiology of Merino Sheep Eds CM Oldham, GB Martin and IW Purvis University of Western Australia, p 59

Tomes GL Robertson DE and Lightfoot RJ (eds) (1979) Sheep Breeding Butterworths, London

[1] Booroolas are a strain of medium-wool Merino with very high lambing rates due to the effect of a major gene - the Fec^B gene

Sheep Health & Production

Chapter 8. Controlled breeding; artificial insemination (ai) and moet

Introduction