Frozen storage of semen

female tract resulting in asynchrony between the age of the spermatozoa and ova Ž . Salamon et al., 1979 . Thus, there was a steeper decline with age of semen in embryonic survival after single than after double insemination, pointing to the impor- tance of time of semen deposition into the cervix to avoid additional ageing of spermatozoa. The problem of low fertility after cervical insemination with liquid stored ram semen still remains unresolved. Increased depth of cervical insemination may improve the fertility, but it has so far been problematic in achieving sufficient penetration and obtaining acceptably high conception rates. A number of attempts have also been made Ž to penetrate the cervical canal and to deposit the semen in the uterus see Sections 3 and . 3.6 . Improvement of the techniques proposed for transcervical insemination and further research in this area seem warranted. The fertility of liquid stored semen can be improved by carefully timed intrauterine insemination by laparoscopy, and when semen containing antioxidants is used. Surgical uterine insemination after laparotomy resulted in acceptable fertility with chilled semen stored for 8 days, and some spermatozoa retained their fertilising capacity for up to 10 Ž . Ž . days Salamon et al., 1979 . Several antioxidants, such as superoxide dismutase SOD , Ž . catalase CAT , cytochrome c and glutathione peroxidase had beneficial effects on maintenance of motility and acrosome integrity of spermatozoa during chilled storage when added to the storage diluent. In fertility tests, using laparoscopic insemination, Ž . Ž . SOD 800 Urml and CAT 200 Urml added to tris–glucose–egg yolk diluent improved the fertility and extended the ‘‘useful’’ storage time to 14 days, when egg Ž fertilisation and pregnancy rates of 50 and 33 have been obtained Maxwell and . Stojanov, 1996 . Storage of semen packaged in encapsulated form, as proposed for bovine semen by Ž . Ž . Nebel et al. 1993 , has been examined in the ram by Maxwell et al. 1996b . After 8 days storage at 58C, the viability and acrosome integrity of ram spermatozoa was lower Ž . Ž for encapsulated in D -lysine and poly D -lysine microcapsules than for control unen- . capsulated semen. In a fertility test, using semen stored at 58C for 20 h for intrauterine insemination, there was no difference between encapsulated and control semen regarding Ž . fertilisation rates 67 of 33 vs. 79 of 44 . Improvement of the viability of encapsulated spermatozoa and attempts to use micro-encapsulated ram semen for cervical insemination remain future tasks.

3. Frozen storage of semen

Ž . The first records on freezing ram semen are by Bernstein and Petropavlovsky 1937 Ž . Ž who successfully used glycerol solution 1 M, 9.2 for storage of mammalian rabbit, . Ž . guinea pig, ram, bull, boar, stallion and avian fowl and duck spermatozoa at y218C; Ž . glycerol at 18 concentration had a toxic effect on spermatozoa. Smirnov 1949, 1950 Ž . Ž during 1947–1950 investigated vitrification without glycerol of small volumes 0.05– . 0.10 ml of ram and bull semen; in a fertility trial in 1949, of 19 ewes receiving three to Ž . five inseminations with frozen semen held for 15 min at y788C , seven ewes produced 11 lambs. In 1951, insemination of 11 cows resulted in the birth of five calves. The Ž . cryoprotective property of glycerol was brought into prominence by Polge et al. 1949 whose finding gave a big impetus to research on cryopreservation of mammalian semen. Considerable experimentation was conducted on frozen storage of ram semen during the 1950s. Initially the method and diluent for freezing bull semen was applied to ram Ž semen with poor fertility results 5 lambing: Blackshaw and Emmens, 1953; Dauzier, . 1956 . It became obvious that the ‘‘bull method’’ had limitations when adopted for ram semen. Over the years of research, a wealth of information has been accumulated on aspects of frozen storage of ram semen such as various diluents, cryoprotective agents, processing, freezing and thawing methods, cryogenic damage and fertility of ram spermatozoa. These aspects will be discussed in the sections that follow. 3.1. Diluents The diluents used for preservation of ram semen, as for other species, generally should have adequate pH and buffering capacity, suitable osmolality, and should protect spermatozoa from cryogenic injury. The diluents discussed here have been grouped on the basis of their chronological use or development, with observations given on their efficiency with regard to fertility. 3.1.1. Citrate–sugar-based diluents Opinions of investigators differed on the ‘‘suitable’’ type of sugar to be included in citrate medium. Using the post-thawing survival of spermatozoa as the criterion, some investigators have chosen arabinose, others fructose or glucose, as components of the citrate diluent. Due to the decrease in osmotic pressure caused by the glycerol in the extender, hypertonic citrate–glucose–yolk or citrate–fructose–yolk diluents with an osmotic pressure of 8–12 atm were generally used. Ram semen, with a mean osmotic pressure of 7.7 atm, tolerated well the above range in diluent tonicity. Also, ram spermatozoa can tolerate a glucose or fructose concentration twice that of isotonicity due to the capacity of monosaccharides to permeate the sperm cells, and thus equalise the osmotic gradient. Ž . Ž The lambing results for semen frozen in ampoules with citrate–glucose or . fructose –yolk diluents in experiments conducted under controlled conditions varied Ž between 17 and 40 after one to three inseminations reviewed by Salamon and . Maxwell, 1995a . The relatively high lambing rates claimed by some workers in the former Soviet Union could not be confirmed and were severely questioned on the basis Ž . of experimental artefacts Lopyrin, 1971 . The citrate–sugar-based diluents were seldom used for freezing ram semen after the late 1960s. 3.1.2. Milk diluents Milk has been adapted for freezing ram semen, mostly in reconstituted form, combined with arabinose, fructose or egg yolk. In comparative laboratory studies, some workers found that pasteurised whole or reconstituted skim milk, citrate–yolk, milk– glucose–yolk and citrate–glucose–yolk were of equal value. Other investigators re- ported better post-thaw sperm survival after freezing with reconstituted skim milk than citrate–yolk, fructose- or lactose-based diluents. Addition of egg yolk to heated ho- mogenised milk did not increase post-thaw sperm survival. Freezing in homogenised milk resulted in better recovery of spermatozoa than when frozen in Norman–Johnson Ž . Ž . N-J-1 and N-J-2 media Patt and Nath, 1969 , but not when frozen in test-yolk, which also prevented the leakage of glutamic oxaloacetic transaminase from spermatozoa Ž . Hinshelwood et al., 1980 . In some laboratory studies, milk preparations were included in synthetic diluents containing sugars and electrolytes. Ž . Ž . The INRA-patented milk–citrate medium was used by several mainly French workers as a glycerolated portion of the two-step dilution method, and was added to the Ž . cooled and partially diluted with lactose–yolk semen. Skim milk has been used as both portions of the two-step dilution, and freezing of semen for practical use in Sweden Ž . Soderquist et al., 1996 . ¨ The fertility results after cervical insemination with milk-frozen semen have been Ž . Ž . variable reviewed by Salamon and Maxwell, 1995a . Some were poor 0–23 , others Ž . Ž . moderate 30–45 . Reports on lambing rates of 50–75 or higher should be regarded with caution. 3.1.3. Lactose-based diluents Successful use of lactose as the main component of diluents for freezing bull semen stimulated its application for ram semen. The initial in vitro studies with lactose-based diluents gave contradictory results, and the first fertility tests were also not convincing. In subsequent tests, the lambing results for lactose–yolk frozen semen were poor Ž . Ž . 12–29 or moderate 40–50 . Lactose–yolk was used for both the non-glycerolated and glycerolated diluent portions, or only for the non-glycerolated portion followed by glycerolated INRA medium. Ž . Disaccharides lactose and saccharose were found more effective in lowering the crystallisation temperature during freezing than monosaccharides, and this was increased Ž . when they were combined with EDTA–Na and diethylamine Platov, 1988 . Inclusion 2 of gum arabic, a complex polysaccharide, in a lactose-based diluent has also been Ž . Ž . examined Platov, 1977 . Gum arabic is a high molecular weight 37,000 , water- soluble substance secreted by acacia plants, and at 1.5–15.0 concentration in the diluent, is well tolerated by spermatozoa and has extracellular cryoprotective properties. Ž . In the studies of Platov 1977 , the semen was diluted in two steps after collection, first with non-glycerolated lactose–yolk and then, after mixing, with lactose–gum arabic– yolk–glycerol or with lactose–yolk–EDTA–Na -diethylamine–gum arabic–glycerol. 2 In further studies, for practical application, Platov et al. elaborated and recommended Ž . the so-called VNII-plem system Russian Animal Research Institute, Lesnie Poljany for Ž . processing and freezing of ram semen Platov, 1988 . According to this system, the semen is diluted in two steps: first, 1.5- to 2-fold with non-glycerolated extender at 288C; second, 2-fold at 248C with glycerolated medium. For both steps of dilution, either Ž . Ž . Ž . Ž . Ž . lactose 9 –dextran 5 –EDTA–Na 0.135 –tris 0.105 –yolk 20 q 2 Ž . Ž . antibiotics q14 glycerol for the second step is used Variant 1 , or lactose Ž . Ž . Ž . 12 –yolk 20 q antibiotics for the first step and glycerolated 17 lactose Ž . Ž . Ž . Ž . Ž . 14.5 –gum arabic 6 –tris 0.6 –citrate 0.27 –yolk 20 q antibiotics for the Ž . Ž . second step Variant 2 . The semen is cooled to 28C in 2 h, then pelleted 0.2 ml on dry ice or a polymer plate at y808C to y908C. In fertility tests comparing the diluent variants of Platov, the claimed lambing results varied from 26 to 60. Ž . Ž Arabonic acid extracted from tree gums substituted for gum arabic Sadykov et al., . Ž 1985 and inclusion of 5–6 polyglukine or 10 reopolyglukine glucose polymers . Ž . with high molecular weight: 30–60,000 in lactose–yolk Zheltobjuk et al., 1981 did not give better fertility than Platov’s diluents. However, insemination with semen frozen in lactose–dextran-based diluents resulted in more acceptable lambing rates. 3.1.4. Saccharose-based diluents Saccharose was used as the main component of synthetic diluents because it was found to protect the acrosome integrity of spermatozoa better than glucose, fructose or Ž . lactose Milovanov and Sokolovskaja, 1980b . Synthetic antioxidants have generally been added to saccharose diluents to inhibit the peroxidation of sperm phospholipids, particularly of unsaturated fatty acids. Peroxidation may be prevented by processing in Ž . anaerobic conditions, and inclusion of antioxidants and chelating agents e.g. EDTA in Ž . the diluent. According to Nauk 1991 , lipid peroxidation, as measured by the malon- Ž . dialdehyde MDA; a secondary peroxide product content of thawed semen, is not Ž . prevented by the freeze–thawing procedure. However, Erochin and Derjazencev 1991 reported that MDA content was similar in fresh-diluted and thawed ram semen, and it increased only during post-thawing incubation at 378C. A substantial amount of work on the inhibition of peroxidation in saccharose-based diluents has been done by workers in the former Soviet Union. Inhibition of peroxida- Ž . tion by providing anaerobic conditions hydrogen or nitrogen atmosphere during processing of semen for freezing proved effective, but technically difficult. Therefore, attention was focused on the use of antioxidants, first of natural or synthetic tocopherol Ž . vitamin E . Tocopherol was most effective when included in saccharide-based diluents containing glucose, EDTA–Na and tris, and when the semen was processed in 2 anaerobic conditions. Subsequently, the diluent was replaced by a saccharose–EDTA– Ž CaNa complex medium EDTA–Na saturated with Ca ions; Milovanov and 2 2 . Sokolovskaja, 1980a , and several synthetic antioxidants were examined such as buty- Ž . Ž . Ž . lated hydroxytoluene BHT , 6-ditrat-butyl-1,4-kresol ‘‘ionol’’ or ‘‘topanol’’ DTBK , Ž . Ž monoethanolamine ‘‘kolamine’’ , phosphoethanolamine, and echinochrome A an ex- . tract from sea urchins with activity 10 times higher than DTBK . Most workers claimed that processing and freezing of semen in diluents containing the above antioxidants improved lambing rates after cervical insemination. Other investigators, however, reported that BHT and vitamin E gave little protection to ram spermatozoa, although it was found that BHT could stabilise the membrane phospho- Ž . lipids and decrease the permeability of the sperm membrane Hammerstedt et al., 1978 , Ž . and that DTBK has a cryoprotective effect Milovanov et al., 1985 . Several variants of the saccharose-based diluents served as a basis of the so-called Ž . VIZ system All-Union Animal Research Institute, Moscow; Milovanov et al., 1985 . The features of this system, recommended by the authors for practical use were: 3- to Ž . 4-fold dilution of semen at room temperature with saccharose 9.8 –EDTA–CaNa 2 Ž . Ž . Ž . Ž . 0.84 –DTBK antioxidant 0.5 –egg yolk 10 –glycerol 5 q antibiotics, cool- Ž . Ž . ing to 2–48C in 3 h, and pellet freezing 0.2 ml on a polymer plate or dry ice . The claimed lambing rates for semen frozen in the saccharose-based diluents with anti- oxidants varied from 19 to 55. Ž Saccharose-based diluents were used also in the KazNITIO system Kasakh Research . and Technological Institute for Sheep Breeding, Alma-Ata; Kasymov, 1984 and the Ž KirgNPOZ system Kirghiz Association for Animal Research and Production; Sadykov . et al., 1985 . The composition of diluents used in the above two systems are described Ž . by Salamon and Maxwell 1995a . 3.1.5. Raffinose-based diluents Raffinose as a diluent component was brought into prominence by the observation of Ž . Nagase and Graham 1964 with bull semen, that sugars of high molecular weight provide better protection to spermatozoa during fast freezing than do those of low molecular weight. The better cryoprotective effect of trisaccharides in comparison to Ž . mono- and disaccharides has also been reported when freezing ram semen. Nauk 1991 found the order of efficiency in stabilising the protein–lipid complex of the cell membrane to be: raffinose saccharose lactose fructose glucose, and the protec- tive effect of sugars in the higher rank was better when they were combined in certain proportions with other sugars, than when used alone. Comprehensive studies on combinations of sodium citrate with various concentrations Ž . of sugars arabinose, glucose, lactose, raffinose led to the elaboration of the raffinose Ž . Ž . Ž . Ž . 9.9 –citrate 2H O; 2 –egg yolk 15 –glycerol 5 medium for pellet freezing 2 Ž . of ram semen Salamon and Lightfoot, 1969; Lightfoot and Salamon, 1969a . These workers found that the optimum osmotic pressure varied with the type of diluent, and that hypertonic media were required for successful freezing of ram semen by the pellet method. The optimum osmotic pressure for the raffinose–citrate–yolk diluent was 375–485 mosM. The high osmotic pressures due to increased raffinose concentrations were less harmful to the survival of spermatozoa than high citrate concentrations. The lambing results were 40–50 after cervical inseminations with semen frozen in this Ž diluent using 150–180 million motile sperm for single and double insemination; . Lightfoot and Salamon, 1970a,b; Salamon and Lightfoot, 1970 . Inclusion of glutamic acid and methionine in raffinose–citrate–yolk resulted in Ž modest lambing after both single and double insemination 31.3 and 39.8; Smirnov . et al., 1978 , and lambing was not improved when tris buffer was added to a Ž . raffinose–glucose–saccharose diluent Ivakhnenko and Aibasov, 1982 . However, it was Ž . claimed Ostashko and Kantsedal, 1976 that, as with bull semen, addition of the Ž . lysomal ferment b-glucuronidase 150 Urml to raffinose–citrate–yolk improved post- Ž . thawing survival and fertility of pellet-frozen ram semen 54.3 vs. 40.8 control . The Ž . lambing was also improved in comparison to the control 37 when peroxidation was Ž . inhibited by processing the semen in a hydrogen atmosphere 44 and also in the Ž . presence of an antioxidant 48 in the combined raffinose–lactose–fructose–mag- Ž . nesium–yolk medium Nauk, 1991 . Ž Raffinose formed the basis of the so-called VNIIOK freezing system All-Union . Sheep and Goat Research Institute, Stavropol elaborated and recommended for practical Ž . use by Zheltobrjuk et al. 1987 . The system consisted of 2.5- to 3-fold dilution of Ž . Ž . semen at 25–308C with a diluent containing raffinose 15 –saccharose 3 –glucose Ž . Ž . Ž . Ž . 0.5 –PVP 3, molecular weight 8000–20,000 –tris 0.07 –yolk 20 –glycerol Ž . Ž . 4.2 q antibiotics, cooling to 2–48C in 3–4 h before pellet freezing 0.15–0.20 ml on Ž . a polymer plate y858C to y958C . 3.1.6. Tris-based diluents Tris in diluents for freezing ram semen was reported by several workers in the early 1970s. Systematic examination of tris as a basic diluent component for pellet freezing Ž . was undertaken by Salamon and Visser 1972 . In their studies, ram spermatozoa tolerated a range of tris concentrations from 250 to 400 mM, and glucose was a more suitable sugar component in the tris medium than fructose, lactose or raffinose. In Ž . Ž . Ž . fertility tests, semen diluted 5-fold with tris 300 mM –glucose 27.75 mM –citric Ž . Ž . Ž . acid 94.7 mM –egg yolk 15 –glycerol 5 q antibiotics and pellet-frozen on dry Ž ice, yielded lambing rates from 30–57 after cervical insemination Visser and Sala- . mon, 1973, 1974a . Ž . Fischer 1990 found that tris with an osmolality of 375 mosMrkg containing 2 egg yolk was the best in preserving acrosomal integrity and motility after thawing. Ž . Combination of 30–290 mM sugars mono- and disaccharides with 100–300 mM tris Ž . maintaining 325 mosmol osmolality did not influence post-thawing motility and Ž . fertility of spermatozoa Molinia et al., 1994 . However, there are also reports that tris performed better in vitro when combined with lactose rather than glucose, saccharose or Ž . milk Zamfirescu et al., 1980 , although tris q skim milk q orvus-es-paste offered some Ž . protection to acrosome integrity Tekin and Gunzel, 1986 . ¨ w Ž . In studies in vitro, Triladil a tris-based diluent proved to be of equal value or a better freezing medium than lactose–yolk and saccharose–lactose–yolk, and addition of 2 bovine serum albumin to Triladil w improved its protective effect on acrosome integrity. However, after insemination the conception rate was better for semen frozen in w Ž . saccharose-based medium than in Triladil Marinov et al., 1983 . Subsequently, w Ž Triladil -frozen semen yielded promising fertility after transcervical insemination Hal- . bert et al., 1990 . Tris–fructose was used as the first diluent part in the two-step method of Fiser et al. Ž . 1987 , and tris has also been included as a component in some lactose- and saccharose- Ž . based diluents. The tris–glucose diluent elaborated by Salamon and Visser 1972 is Ž widely used and recommended for the frozen storage of ram semen Salamon, 1976; . Evans and Maxwell, 1987 . 3.1.7. Other diluents Zwitterion buffers such as tes, hepes and pipes have been used with varying success as the basis of diluents for freezing ram semen. Fertility of semen frozen in test medium Ž . Ž . tes titrated with tris varied from poor to satisfactory 13–67 lambing . Test combined with skim milk and the zwitterion buffers tes, hepes and pipes were better than tris–glucose–yolk diluents in terms of post-thawing motility, but inferior in preserving acrosomal integrity of spermatozoa. Fertility was lower for semen frozen in zwitterion buffers than in tris–glucose–yolk. Noteworthy among other substances examined are dextran and hydroxyethyl starch. Ž When these were added to a combined medium tes–citrate–glycine–lactose– . raffinose–fructose–citric acid and used as the non-glycerolated diluent portion, fol- lowed by glycerolated milk–citrate, more effective cryoprotection was provided to spermatozoa than by using lactose–yolk as the first and INRA medium as the second Ž . diluent portion Fiser, 1975 . 3.2. ProtectiÕe agents and methods of addition to semen 3.2.1. Glycerol Glycerol is the most commonly used protective substance in diluents for freezing ram semen. For semen frozen by the slow ‘‘conventional’’ method, and using mainly hypertonic diluents, most investigators found that the optimal glycerol concentration was within the range of 6–8; spermatozoa frozen rapidly by the pellet method survived Ž . best with 3–4 glycerol in the diluent. Graham et al. 1978 reported that glycerol levels above 6 were detrimental to post-thawing survival of spermatozoa. The action of glycerol on ram and bull spermatozoa seems to be different, as it more easily Ž . penetrates ram spermatozoa Nauk et al., 1970 . It was suggested that the optimal glycerol concentration in diluted semen is also related to its final concentration relative Ž . to the spermatozoa Colas, 1975 . The level of glycerol included in diluents for frozen storage of ram semen is Ž . ultimately limited by its toxicity Fahy, 1986 , which in turn depends on cooling and freezing rate, diluent composition, and method of addition of glycerol. Examination of Ž the combined effects of glycerol concentrations and cooling rates 0–8 and pelleting on dry ice and into liquid nitrogen, Visser and Salamon, 1974c; 0–16 and cooling at . 1–1008Crmin, Fiser and Fairfull, 1984 showed that the higher the cooling rate, the lower the optimum glycerol concentration, and the best post-thaw sperm survival rates observed by the latter investigators were for 4–6 glycerol and a freezing rate of 10–1008Crmin. Apart from the cooling rate, the optimum glycerol concentration also depends on the Ž composition of the diluent and in particular on its osmotic pressure Salamon, 1968; . Lightfoot and Salamon, 1969a . Furthermore, the glycerol concentration may be influ- enced by the egg yolk level in the diluent, as increased concentrations of egg yolk may Ž . reduce the required concentration of glycerol Watson and Martin, 1974 . There are reports indicating that inclusion of antioxidants with a cryoprotective effect in the diluent may also allow the use of a relatively low glycerol concentration of 3.5, Ž . 2.5 or 2.0 Varnavskij and Varnavskaja, 1976; Milovanov et al., 1985 . Ž . Glycerol can be added to the semen in a separate diluent fraction two-step dilution , Ž . or by a single addition of the diluent containing glycerol one-step method . Initially, it was found better to add the glycerolated diluent portion at 298C than at 58C, but in subsequent tests addition at 58C was more suitable. Addition of glycerolated diluent at 4–58C was preferred by most workers, but some found no difference between addition at either 328C or 38C or at 228C or 58C. The glycerol-containing diluent generally was of the same type as that used for initial Ž . extension at 308C , but media of differing composition have also been used. French workers found that post-thawing recovery and fertility of spermatozoa were better when the partially diluted semen was glycerolated with INRA medium than with the initial lactose–yolk extender, and lambing was also better when glycerolisation occurred at 48C Ž . rather than at 308C Colas, 1975; Colas and Brice, 1975 . Ž . Colas 1975 suggested that glycerol may be slightly toxic to spermatozoa even at a concentration of 4 and its harmful effect is less when added at a temperature close to 08C. Early workers using two-step dilution recommended a gradual addition of the glycerol-containing diluent at 2–58C. However, it has been found that single addition of Ž . the glycerolated portion before freezing at 58C was as effective, or better than, the gradual addition in divided portions. Ž . Contrary to the work recommending the use of two-step dilution, Salamon 1968 and Ž . Lightfoot and Salamon 1969a reported that a single dilution of semen with glycerol- Ž . containing diluent at 308C ‘‘one-step’’ method was as good as two-step dilution, but the efficiency of the former method depended on the sugar component of the diluent. One-step addition of glycerol at 308C is a practical and widely used method for dilution Ž . of ram semen for frozen storage Salamon, 1976; Evans and Maxwell, 1987 . While glycerol offers cryoprotection to spermatozoa, it may also cause structural damage during pre-freeze processing. Consequently, it was suggested that glycerol should be added no more than 20–30 min before freezing. Effective cryoprotection after Ž . short 5–10 s contact with glycerol has been demonstrated for bull and boar, and also Ž . for ram semen 0–5 min , which supports the earlier view that penetration of glycerol into the cell is not essential for protection. Removal of glycerol from thawed semen by centrifugation or by dialysis had no effect on lambing. Successes in obtaining fertility with ram semen frozen without glycerol have been Ž . Ž . reported by Lopatko 1976 and Abdelhakeam et al. 1991a who claimed 26 and 52 lambing rates, respectively. For freezing ram semen without glycerol, Abdelhakeam et Ž . al. 1991b diluted the semen at 58C, 3 h after collection, with hypertonic test buffer containing 25–30 egg yolk and 10 maltose monohydrate. The efficacy of the procedure remains to be confirmed. Several agents other than glycerol have also been examined for their cryoprotective Ž . action on ram spermatozoa. Such agents were dimethylsulfoxide DMSO , ethylene glycol, albumin, low molecular weight polyols, polymeric compounds, surfactants, high Ž concentrations of sugars of various types, compatible solutes proline, glycine betaine, . Ž taurine and antifreeze proteins from polar fish reviewed by Salamon and Maxwell, . 1995a . None of the agents examined proved better than glycerol. 3.2.2. Egg yolk Egg yolk is a common constituent of semen diluents, protects the spermatozoa against cold shock and confers protection during freezing and thawing. It is believed to act at the level of the cell membrane, and to have a bigger effect for bull than ram spermatozoa. A wide range of egg yolk concentrations have been examined in diluents for freezing of ram semen. Early workers used 30–50, but most subsequent investigators included lower concentrations in the diluent. For freezing ram semen in ampoules, egg yolk concentrations of 3–6 seemed sufficient, but for pellet freezing 15 was the optimum Ž concentration, although the effect depended on the composition of the diluent Salamon . and Lightfoot, 1969 . Some investigators examined egg yolk concentrations as low as Ž . 1.5–3.75, but others Graham et al., 1978 found that low levels were unsatisfactory. Ž . The saccharose–EDTA–CaNa complex diluent allowed the use of low 4–5 egg 2 Ž . yolk concentrations and even its elimination was suggested Milovanov et al., 1985 . On Ž . the other hand, in the attempt of Abdelhakeam et al. 1991b to freeze ram semen without glycerol, it was necessary to increase the egg yolk to 25–30 in the test diluent for successful freezing. Ž . Egg yolk, although sometimes partially replaced by substance s with similar activity, is still likely to remain an important component of diluents for freezing ram semen, particularly due to its protective effect on the plasma membrane. 3.3. Processing and freezing of semen The success of deep freezing depends to a notable degree on the rate of dilution of semen. Originally, semen was diluted to protect spermatozoa during cooling, freezing and thawing, but the rate of dilution was often changed for technical reasons, such as to increase the number of females which could be inseminated with each ejaculate, or to standardise the number of spermatozoa in each dose of frozen–thawed semen. The pre-freezing dilution rate used by early workers varied from 11- to 26-fold, but Ž . the majority of subsequent investigators used 2- to 6-fold vrv dilution rates. As the diluent composition was not adjusted for the different rates of dilution, it could not be determined whether any effect on spermatozoa was due to dilution per se, or to variation of the diluent components in the diluted semen. For freezing ram semen nowadays, Ž . generally 2- to 5-fold vrv pre-freezing dilution rates are used, with the diluent Ž composition adjusted for the rate of dilution Salamon, 1976; Evans and Maxwell, . 1987 . After dilution, the semen is cooled to a temperature close to 08C. Cooling is a period of adaptation of spermatozoa to reduced metabolism. Traditionally, equilibration has been regarded as the total time spermatozoa remain in contact with glycerol before freezing, during which it penetrates into the sperm cell to establish a balanced intra- cellular and extracellular concentration. It should not be overlooked that the term ‘‘equilibration’’ includes the concentration balance not only of glycerol, but also of the other osmotically active diluent components. Glycerol has been implicated in some undesirable phenomena occurring during equilibration such as changes in the structure and biochemical integrity of spermatozoa and acceleration of the acrosome reaction. Although these may be overlooked in the light of the other favourable cryogenic properties of glycerol, it should also be noted that this medium may, to a certain extent, adversely affect the fertility. With the two-step method of dilution the contact with glycerol begins at 2–58C. However, when one-step dilution is adopted, glycerolisation begins at 308C. In both methods, a period of cooling to 2–58C is required, the duration of which generally varies from 1 to 3 h. Rapid cooling of extended semen from 308C to about 158C may have no effect on survival of spermatozoa, but fast cooling from 308C to 108C, 58C or 08C Ž . decreases the post-thaw motility of spermatozoa Fiser and Fairfull, 1986 . In the case of one-step dilution to the final rate at 308C with the glycerolated Ž . medium, a combined cooling–equilibration storage is involved. In the studies on pellet Ž . freezing of ram semen Lightfoot and Salamon, 1969a , the optimum period of storage Ž . equilibration after one-step dilution and cooling to 58C depended on the sugar composition of the diluent and the glycerol concentration. The presence of raffinose in the diluent allowed a decrease in equilibration from 2.5 to 1 h. Nowadays the one-step method of dilution with glycerolated medium is generally used and it is recommended to Ž freeze the semen after 1.5 to 2 h cooling to 58C Salamon, 1976, Evans and Maxwell, . 1987 . There seems to be no convincing evidence supporting either a longer coolingrequilibration period, or the need for the more complicated two-step dilution procedure. The cooling rate of diluted semen from temperatures just above 08C can significantly influence the survival of spermatozoa after freezing. Ram semen frozen in ampoules has generally been cooled at the slow ‘‘conventional’’ rate applied for bull semen. This method is seldom used today, and freezing is done in PVC straws and minitubes or in pellet form. Freezing of semen in straws is performed by suspension in liquid nitrogen vapour, and the velocity of cooling is regulated by the distance of the straws from the level of the liquid nitrogen. Ram spermatozoa tolerate a range of freezing velocities in straws. Thus, suspension of straws in liquid nitrogen vapour between y758C and y1258C had Ž . no effect, but vapour at y558C reduced the survival of spermatozoa Colas, 1975 . Although cooling rate was found to interact with glycerol concentration, the cooling rates from 10 to 1008Crmin had a smaller effect on the survival of spermatozoa than Ž . glycerol concentration Visser and Salamon, 1974c; Fiser and Fairfull, 1984 . The shape of the freezing curve may also be important for straw freezing. Instead of a linear decrease in temperature, it is better to cool the semen according to a parabola- shaped curve, which can be achieved by suspending the straws 4–6 cm above the liquid nitrogen surface. Post-thaw recovery of motility of spermatozoa frozen in straws and minitubes on the surface of dry ice was poorer than of pelleted spermatozoa, but this Ž . was not reflected in the fertility rate Maxwell et al., 1995a . Pellet-freezing is generally done by placing semen droplets into depressions on dry Ž . ice or on a polymer plate cooled to y808C to y958C in the former Soviet Union . The velocity of cooling can be regulated by the volume of the semen pellet and the temperature of the freezing agent. Nevertheless, pellet volumes from 0.03 to 0.86 ml, with different surface to volume ratios, have been frozen on dry ice without a reduction Ž . in post-thaw motility, although the cooling rates varied 95 to 308Crmin for pellet sizes Ž . and also within the pellet Lightfoot and Salamon, 1969b; Visser and Salamon, 1974b . Increased cooling rates obtained by pelleting on a stainless steel plate or on dry ice Ž . cooled in liquid nitrogen y1008C to y1608C had no effect on recovery of sperm Ž . motility Salamon, 1970 and lambing for semen pellet-frozen at y798C or y1408C Ž . was not different Salamon, 1971 . Dropping the semen directly into liquid nitrogen resulted in poor recovery of motility, or all spermatozoa died. The reason for this may be that liquid nitrogen is not a good medium for very fast freezing. Since it is a liquid at its boiling point, it has a calefactory effect which delays the thermal contact with the Ž . Ž . semen Rey, 1957 . However, when semen droplets 0.03 ml were pipetted into liquid Ž . nitrogen, or vials with semen 1.5–2.5 ml were immersed into liquid nitrogen for 5–7 s, then elevated and held in liquid nitrogen vapour for a few minutes before re-immersion into liquid nitrogen, post-thaw motility of pelleted spermatozoa was nearly similar to Ž . that of sperm pelleted onto dry ice Visser and Salamon, 1974c , and after insemination Ž . with the semen thawed in vials satisfactory pregnancy rates 51–69 were claimed Ž . Kasymov and Ashimov, 1975 . 3.4. Thawing of semen In the freeze–thaw procedure, the warming phase is just as important to the survival of spermatozoa as the cooling phase. Spermatozoa that have survived cooling to y1968C still face the challenge of warming and thawing, and thus must traverse twice Ž . the critical temperature zone y158C to y608C . Both rate of cooling and thawing exert an effect on the survival of spermatozoa Ž . Mazur, 1985 . The effect depends on whether the rate of cooling has been sufficiently high to induce intracellular freezing, or low enough to produce cell dehydration. In the former case, fast thawing is required to prevent recrystallisation of any intracellular ice present in the spermatozoa. Spermatozoa thawed at a fast rate may also be exposed for a Ž . shorter time to the concentrated solute and cryoprotectant glycerol , and the restoration of the intracellular and extracellular equilibrium is more rapid than for slow thawing. There was no agreement among early investigators on the optimum temperature for thawing of semen frozen in ampoules. Some found that ram spermatozoa cooled slowly in ampoules by ‘‘the bull method’’ revived better when thawed at 40–438C, than slowly ‘‘by reversing the cooling procedure’’. Other workers thawed the ampoules in ice water, Ž . in water at 2–58C, 6–88C or at room temperature 18–208C with conflicting results regarding recovery of spermatozoa. In the majority of reports, ampoule–frozen ram semen was thawed in a water bath at 378C. Ram semen frozen in straws has been thawed by most investigators at 38–428C. Ž . Some workers reported that thawing at high temperatures 60–758C was comparable to that at 38–428C in terms of post-thaw motility, acrosome integrity and fertility of spermatozoa. Ž . Pellet-frozen ram semen may be thawed either in a solution wet thawing or in dry Ž . tubes dry thawing . In the former case, not only the presence, but also the composition of the thawing solution influenced the recovery of sperm motility, and the effect Ž depended on the composition of the sugar–citrate–yolk freezing diluent Lightfoot and . Salamon, 1969b . Inositol–citrate, glucose–citrate and fructose solutions were the best Ž . and of equal value for thawing at 378C semen pellets frozen in raffinose–citrate–yolk diluent. A relationship between composition of freezing diluent and of thawing solution Ž was also found when the tris-based freezing media were used Salamon and Visser, . 1972 . Further, there were interactions between the composition of freezing diluents and Ž . method of dry or wet thawing. Thawing temperatures between 378C and 758C have been examined for both wet and dry thawing, and generally improvements in post-thaw motility rates were observed with increasing temperature. In order to maintain the benefit of a high rate of warming, without the risk of overheating the semen, interrupted thawing or the use of special thawing devices have been recommended. In the former case the semen pellet, placed in Ž . warm thawing solution 60–808C , was transferred to a water bath at 35–408C when Ž . about two-thirds of the pellet has melted Smirnov et al., 1978 . The thawing devices assure a continuous and fast separation of the liquid portion from the remaining solid pellet, and thus prevent further heating of the thawed semen portion. The different types Ž . of thawing device have been described by Salamon and Maxwell 1995a . 3.5. Causes of low fertility after cerÕical insemination Satisfactory sperm survival rates were obtained with a number of diluents and the complex technology of freezing ram semen appears to be satisfactorily developed. Nevertheless, final judgement on the technologies elaborated should be tempered, because lambing results similar to those for fresh semen have only rarely been obtained after cervical insemination. The low fertility after cervical insemination with frozen– thawed ram semen could be due to a variety of factors. 3.5.1. Damage to spermatozoa during freeze–thawing Ž . Although a relatively high proportion 40–60 of ram spermatozoa preserve their motility after freeze–thawing, only about 20–30 remain biologically undamaged. A spermatozoon may be motile, but damaged, in which case it is doubtful if such a cell will fertilise the egg. Motility and structure of the spermatozoa are affected to different extents, and it is not known whether the changes occur simultaneously or are caused at different stages of the freeze–thaw procedure. Ž . The basic cryogenic damage to spermatozoa may be ultrastructural physical , biochemical or functional. Ultrastructural damage occurs to the plasma and acrosome membranes, the acrosome, the mitochondrial sheath and the axoneme. Ultrastructural damage generally is more severe for ram than bull spermatozoa. After both slow and fast freezing of ram semen, motility is better preserved than the morphological integrity of spermatozoa. The plasma and acrosome membranes are more sensitive than the nucleus Ž . and locomotor mid-piece part of the sperm cell. The outer membrane of the acrosome is more vulnerable than its inner part and internal membrane. It has been observed that the mitochondrial architecture is altered by freeze–thawing, but the tail filament and fibrils showed no detectable change after freeze–thawing. Ultrastructural damage during freeze–thawing is accompanied by biochemical changes or loss of their vital contents. Some of the measured losses and changes include the Ž . release of glutamic oxaloacetic transaminase GOT , losses of lipoproteins and amino acids, decrease in phosphatase activity, decrease in loosely bound cholesterol protein, increase in sodium and decrease in potassium content, inactivation of hyaluronidase and acrosin enzymes, loss of prostaglandins, reduction of ATP and ADP synthesis, decrease Ž . in acrosomal proteolytic activity reviewed by Salamon and Maxwell, 1995b . Denatura- tion of DNA is also possible, as modifications in the chromatin structure of bull, boar, Ž cat, human and recently of ram spermatozoa have been observed Gillan and Maxwell, . 1999 . These ultrastructural and biochemical cryogenic changes to spermatozoa could be responsible for a decrease in their functional integrity, survival in vivo, and fertilising capacity. 3.5.2. Transport, Õiability and fertilising ability of spermatozoa in the female genital tract Ž A number of investigators have examined these aspects reviewed by Salamon and . Maxwell, 1995b and found that there is inadequate transport and reduced viability of frozen–thawed spermatozoa in the genital tract of the ewe, which are the major causes of low fertility after cervical insemination. After cervical insemination, frozen–thawed spermatozoa were expelled faster than fresh spermatozoa from the female reproductive Ž . tract Gillan and Maxwell, 1999 . However, when the thawed semen was deposited into Ž . the uterus or oviducts, high 85–95 egg fertilisation rates have been obtained, showing that the presumably biologically intact spermatozoa maintained their fertilising capacity after freeze–thawing. 3.5.3. Capacitation Ž . It has been advocated Watson, 1995; Maxwell and Watson, 1996 that some aspects Ž . of the cooling–freezing–thawing cycle similar to liquid storage may advance the maturation of sperm membranes, and increase the proportion of capacitated and acro- some reacted spermatozoa. Changes to the membranes of spermatozoa may not affect motility, but shorten the lifespan of the cells and render them less fertile or incapable of fertilisation. Ageing of capacitated spermatozoa in the reproductive tract after cervical insemination may further aggravate the situation. If such spermatozoa are not exposed to oocytes within a short time after insemination, fertilisation will not occur, as the cells Ž may prematurely die in the lower part of the reproductive tract. In vitro studies Gillan . and Maxwell, 1999 showed that frozen–thawed spermatozoa are released earlier than fresh spermatozoa after binding to oviduct cells, confirming their physiological readiness Ž to participate in fertilisation. The membrane status of spermatozoa intact, capacitated . Ž and acrosome reacted can be assessed by functional assay zona-free hamster egg w x . penetration: Garde et al., 1993; and chlortetracycline CTC staining: Gillan et al., 1997 . Ž . Where hamsters are not available like Australia, due to quarantine restrictions , in vitro Ž . matured zona-intact sheep oocytes may be used for penetrationrfertilization assays Ž . Gillan and Maxwell, 1999 . 3.5.4. Embryonic mortality Spermatozoa weakened or not mortally injured during freeze–thawing, and those which after thawing and insemination are aged in the female tract may initiate embryos Ž . that are not viable and perish at an early stage. Maxwell et al. 1996a found that after in vitro fertilisation of in vitro matured sheep oocytes, the stage of fertilisation may be more advanced for oocytes inseminated with frozen–thawed than fresh spermatozoa. The fast cleavage observed also after cervical insemination with liquid stored semen Ž . Lopyrin and Rabocev, 1968 , can be considered to presage embryonic mortality. There Ž . is some evidence Gillan et al., 1997 that ewes inseminated with frozen–thawed Ž . capacitated spermatozoa into the uterine horns, and at a time close to ovulation are more likely to produce viable embryos. 3.5.5. Other causes of low fertility Late oestrus is not favourable for transport and survival of spermatozoa and fertilisa- tion is generally lower for short than long oestrus. Fertility may be reduced if eCG is not used in combination with a progestagen sponge for synchronisation and induction of Ž . Ž . oestrus, and fertility is also low when oestrus occurs early 36 h rather than late 48 h after progestagen sponge removal. The optimum time of insemination is between 48 and 58 h after sponge removal. 3.6. Methods and attempts to improÕe fertility after cerÕical insemination Since the low fertility obtained after cervical insemination with frozen–thawed semen is primarily due to failure of establishment of an adequate cervical population of functionally intact spermatozoa, which is associated with impaired sperm transport in the ewe’s genital tract, several methods and substances have been used in attempts to improve the situation. 3.6.1. Increased concentration of spermatozoa An obvious method was the reconcentration of the thawed semen by centrifugation in order to deposit high numbers of spermatozoa at insemination. This resulted in an increased number of sperm cells throughout the tract up to the oviduct, improved egg Ž fertilisation and lambing Lightfoot and Salamon, 1970b; Salamon and Lightfoot, 1970; . Colas and Guerin, 1981 . Reconcentration of thawed semen by filtration through a ´ Ž . milliporous membrane Volkov, 1974 and insemination of high numbers of spermato- Ž zoa in increased volumes have also been used to increase fertility Colas and Brice, . 1976; Colas, 1975 . However, the above methods were of low efficiency, as only a few ewes could be inseminated with one frozen–thawed ejaculate. Dilution of semen at a Ž . low rate 1.5-fold before freezing in order to obtain inseminates with a high concentra- tion of thawed spermatozoa yielded poor lambing. 3.6.2. Use of relaxin, oxytocin, prostaglandins, other substances and methods Injection of ewes with relaxin, oxytocin and prostaglandins had two possible aims: to relax the cervix, which would permit deep cervical or uterine deposition of frozen– thawed semen, or to increase the contractility of the genital tract and thus improve the transport of spermatozoa. Relaxin injected 12 h before insemination had no relaxation effect on the cervix and Ž . did not affect the mean depth of insemination Salamon and Lightfoot, 1970 . Oxytocin stimulated the contractile activity of the cervix and uterus in vivo and in vitro, dilated the cervix and improved sperm transport after mating, but not after insemination with frozen–thawed semen in which case lambing was also depressed. Introduction of the muscle relaxant cocaine into the cervix, or electrical stimulation Ž . Ž . 3.7 V of the cervix before insemination to induce uterine contraction , had no Ž . w Ž beneficial effect on lambing Varnavskij and Turbin, 1974 . Injection of Partusisten a . tocolytic substance 20 min before insemination improved the penetrability of the cervix Ž . but not the lambing Nehring et al., 1989 . Ž . Prostaglandin PG E , PGE and PGF a stimulated the contractile activity of the 1 2 2 cervix and uterus in vitro and in vivo, and supplementation of semen with PGE and 1 PGF a before freezing also improved the transport of spermatozoa in the genital tract. 2 PG improved the sperm motility and lambing when added to thawed semen, but not when used before freezing. Injection of ewes with PGF a before insemination with 2 thawed semen also improved the lambing results, and hylase and buscolysin injected Ž intramuscularly increased the penetrability of the cervix reviewed by Salamon and . Maxwell, 1995b . Treatment of frozen–thawed semen with metabolic stimulants such as methylxan- thines caffeine and pentoxifylline, proteinase, kallikrein calcium, cyclic adenosine-3 X , X Ž . 5 -monophosphate cAMP , and 2-deoxyadenosine, which in a number of other species stimulated sperm motility, had no such effect on ram spermatozoa, except for pentoxi- fylline. However, as the increase in motility by the latter compound was not reflected in Ž . improved fertility after intrauterine insemination Maxwell et al., 1995b , it is doubtful whether pentoxifylline would improve the fertility in the case of cervical insemination. 3.6.3. Double insemination Double insemination is a generally accepted method of increasing fertility. The magnitude of the response may depend on the sperm concentration in the inseminate and the timing of insemination in relation to the stage of oestrus. Little advantage was gained from the second insemination when the first had been performed at about the middle of Ž . oestrus Salamon, 1972 . It should also be noted that in studies on double insemination, the number of motile frozen–thawed spermatozoa deposited by two inseminations was twice that deposited by single insemination, and in such a case the benefit of double Ž insemination can be accounted for by the increased number of spermatozoa Visser and . Salamon, 1974a . When equal total numbers of motile spermatozoa were deposited by Ž . single and double inseminations within the optimum time range 12–28 h after detection of oestrus, the lambing results were similar, and the number of motile spermatozoa inseminated was more important than whether single or double insemina- Ž . tions were performed Salamon, 1977 . Regarding the time interval between insemina- tions, a better lambing result can be expected when the first, performed on the morning of detection of oestrus, is followed by the second 8–10 h rather than 24 h later. 3.6.4. Depth of insemination Penetration of the ewe’s cervix is difficult due to its peculiar structure, and for deposition of thawed semen deep into the cervical canal different techniques and devices Ž have been adopted. The cervical traction method which consisted of pulling the . entrance of the cervix into the vagina by forceps and the cervical traction combined Ž . with digital manipulation of the cervix through the rectum Andersen et al., 1973 allowed semen deposition to a depth of 2–5 cm, and the latter method even uterine deposition of semen. Both methods improved the fertility, but were stressful to the animal. Deep cervical semen deposition was easier to achieve by special inseminating Ž . pipettes and devices described by Salamon and Maxwell, 1995b . The lambing results Ž . improved with increased depth of insemination into the cervix Fig. 1 , and were Fig. 1. Effect of depth of insemination on fertility of frozen–thawed ram semen introduced through the cervix Ž . weighted means from 26 publications; 9716 ewes . relatively consistent between workers inseminating to approximately the same depth. Deep cervical insemination allowed a decrease in the number of motile spermatozoa to a dose of 20 or 40 million, which after single insemination gave 50 and 53 lambing respectively, compared with 51 for control insemination with 80–100 million motile Ž . spermatozoa Stojanov, 1980 . 3.6.5. Intrauterine insemination The problem of the cervical barrier in the ewe can be overcome by deposition of Ž . frozen–thawed semen into the uterus via the cervix transcervical insemination or directly into the uterus by laparotomy or laparoscopy. Success of penetration transcervically into the uterus varied between investigators from 39 to 62. By using the ‘‘Guelph system’’ which involved traction of the cervix, Ž . 87 successful penetration has been claimed Buckrell et al., 1992 . The transcervical insemination methods, although offering success for intrauterine insemination, vary regarding the repeatability of penetration, are time consuming and rather stressful to the animal. In an experiment conducted in Australia, the pregnancy rate at day 70 for ewes Ž . inseminated by laparoscopy 48 was higher than for ewes inseminated by transcervi- Ž . Ž . Ž . cal intrauterine 32 or cervical 9 methods Windsor et al., 1994 . Surgical insemination by laparotomy was used by early investigators to examine the fertilising capacity of frozen–thawed spermatozoa. Since the introduction of laparo- Ž . scopic semen deposition Killeen and Caffery, 1982 , this method has been generally Ž adopted for intrauterine insemination in most sheep producing countries Evans, 1991; . Haresign, 1992 , as it offers reliable and predictable lambing results. Noteworthy reports are summarised in Table 1. Laparoscopic insemination has been mainly performed at a synchronised oestrus and to a lesser extent in natural oestrus. In the case of synchronised oestrus, the main variables examined have been type of synchronising device, season of synchronisation, time of insemination, dose of inseminate and site of uterine deposition. 3.6.5.1. Type of synchronising deÕice. Laparoscopic insemination yielded better lambing Ž . in ewes synchronised with fluorogestone acetate FGA; 30 mg Chronogest than with Ž . medroxy-progesterone acetate MAP; 60 mg Repromap sponges, and the time of S. Salamon, W.M.C. Maxwell r Animal Reproduction Science 62 2000 77 – 111 98 Table 1 Summary of noteworthy results on fertility of frozen–thawed semen after intrauterine insemination by laparoscopy Reference Diluent Inseminated Time of No. of ewes Fertility Comments: Ž dose total no. insemination inseminated method of 6 Ž sperm=10 in hours synchronisation: two uterine after sponge sponge, or a b c . . horns removal other fertility data , or Ž . Maxwell 1984 Tris–glucose– 40 motile 60–66 837 50.7 Mean from 17 flocks 2 yolk–glycerol FGA sponge a Salamon et al. Raffinose– 10 motile 60–64 45 57.8 FGA sponge Ž . 1985 citrate– 20 motile 50 64.0 yolk–glycerol 40 motile 48 58.3 80 motile 50 66.0 Eppleston and Tris–glucose– 50 motile 48 41 31.7 Data are presented as Ž . Roberts 1986 yolk–glycerol 60 39 61.5 least-squares 2 72 46 37.0 means MAP sponge 48 41 51.2 60 40 52.5 FGA sponge 72 56 62.5 c Maxwell and Tris–glucose– 20 motile 60 50 54.0 FGA sponge Ž . Barnes 1986 yolk–glycerol w c 50 60.0 CIDR a Ž . Maxwell 1986a Tris–glucose– 20 motile 48 111 45.9 FGA sponge yolk–glycerol 60 107 55.1 72 115 57.4 78 61 39.3 S. Salamon, W.M.C. Maxwell r Animal Reproduction Science 62 2000 77 – 111 99 Ž . a Maxwell 1986b Tris–glucose– 0.5 motile 60 75 29.3 FGA sponge yolk–glycerol 5 motile 164 25.0 10 motile 85 38.8 20 motile 96 53.1 25 motile 87 56.3 50 motile 103 62.1 a 20 motile 75 44.9 FGA sponge Insem. in ipsilateral a 69 76.8 FGA sponge horn only. Insem. in both uterine horns. Findlater and Tris–glucose– NA 48 122 64 Type of sponge used a Ž . Haresign 1987 yolk–glycerol 60 63 not stated 72 44 d e d e b Ž . Hunton et al. 1987 Tris–glucose– NS 59–63 61 r60 49 r53 FGA sponge d e d e yolk–glycerol 38 r38 66 r47 d e d e 38 r39 66 r49 a Ž . Nehring et al. 1989 Milk–fructose– 50 56–60 20 50.0 FGA sponge yolk b Ž . Ž . Walker et al. 1989 Tris–glucose– 40 48–52 or 330 57.3 FGA sponge No GnRH control yolk–glycerol 56–60 86 32.6 GnRH 24 h after removal 326 57.7 GnRH 36 h after removal Gourley and Tris–glucose– 20 55–60 294 60.2 Insemination in breeding Ž . Riese 1990 yolk–glycerol season 30 63.3 FGA sponge Insemination in non- and norgesto- breeding season b met implant continued on next page S. Salamon, W.M.C. Maxwell r Animal Reproduction Science 62 2000 77 – 111 100 Ž . Table 1 continued Reference Diluent Inseminated Time of No. of ewes Fertility Comments: Ž dose total no. insemination inseminated method of 6 Ž sperm=10 in hours synchronisation: two uterine after sponge sponge, or a b c . . horns removal other fertility data , or b Quintana Casares Tris–glucose– 23.3 motile 50 37 40.5 All FGA sponge Ž . et al. 1990 yolk–glycerol 39.5 motile 63 52.4 58.6 motile 77 57.1 77.1 motile 68 57.4 104.5 motile 31 58.1 a Ž . Findlater et al. 1991 Tris–glucose– 10.4 motile 54 44 52 All FGA sponge Semen diluted 4-fold, inse- yolk–glycerol minate dose 80 ml 20.8 motile 54 44 53 Semen diluted 2-fold, inse- minate dose 80 ml 41.6 motile 54 44 56 Semen diluted 3-fold, inseminate dose 80 ml 83.2 motile 54 44 67 Semen diluted 3-fold, inse- minate dose 160 ml 26.0 motile 54 Total 929 54 Alteration of semen dose achieved by increasing inseminated volume from pooled semen from 40 to 160 ml 52.2 motile 54 59 104.4 motile 54 61 41.6 motile 48 35 65 41.6 motile 60 35 63 41.6 motile 72 35 46 52.2 48 Total 999 57 52.2 54 55 52.2 60 58 S. Salamon, W.M.C. Maxwell r Animal Reproduction Science 62 2000 77 – 111 101 Fukui et al. Tris–glucose– 100–160 36–60 75 54.7 MAP sponge a Ž . 1993a yolk–glycerol w a 69 49.3 CIDR Fukui et al. Tris–glucose– 36 44–52 55 56.4 0.05 ml inseminate dose Data pooled for progesterone w a Ž . 1993b yolk–glycerol 71 59 62.7 0.1 ml inseminate dose sponge and CIDR 143 60 55.0 0.2 ml inseminate dose b Windsor et al. Tris–glucose– 60 51–54 251 48 All FGA sponge Laparoscopic intrauterine insem. Ž . 1994 yolk–glycerol 80 51–56 201 32 Transcervical intrauterine insem. 200 54–56 256 9 Cervical insemination b Maxwell et al. Tris–glucose– 20 motile 60 150 70.7 All MAP sponge Semen frozen in pellets Ž . 1995a yolk–glycerol 150 56.7 Semen frozen in 0.25 ml straws 150 55.3 Semen frozen in 0.5 ml straws 150 65.3 Semen frozen in minitubes b Maxwell et al. Tris–glucose– 36 59–63 42 52.4 All MAP sponge Thawed semen treated with PBS Ž . 1995b yolk–glycerol containing pentoxifylline 39 41.0 Thawed semen treated with PBS Ž . control b Ž . Molinia et al. Tris 360 mM – 20 50–60 42 66.7 All FGA sponge Ž . Ž . 1996 glucose 33 mM – yolk–glycerol Ž . Tris 300 mM – 40 47.5 Ž . glucose 30 mM – yolk–glycerol Ž . Hepes 236 mM – 45 71.1 NaOH–glucose– yolk b Gillan et al. Tris–glucose– 30 motile 50–54 107 77.6 All FGA Sponge Semen unincubated; pregnancy Ž . 1997 yolk–glycerol day 18 107 69.2 Semen unincubated; pregnancy day 50 continued on next page S. Salamon, W.M.C. Maxwell r Animal Reproduction Science 62 2000 77 – 111 102 Ž . Table 1 continued Reference Diluent Inseminated Time of No. of ewes Fertility Comments: Ž dose total no. insemination inseminated method of 6 Ž sperm=10 in hours synchronisation: two uterine after sponge sponge, or a b c . . horns removal other fertility data , or 98 71.4 Thawed semen incubated for 6 h; pregnancy day 18 98 51.0 Thawed semen incubated for 6 h; Pregnancy day 50 b Sanchez-Partida Tris–glucose– 30 56–60 39 41.0 All MAP sponge ´ Ž . et al. 1997 yolk–glycerol Tris–glucose– 41 41.4 yolk–glycerol q50mM taurine Ž . Moses et al. 1997 Tris–glucose– 100 786 69.6 All MAP sponge Ewes exhibiting oestrus yolk–glycerol 72–74 195 36.0 Ewes not exhibiting oestrus 179 59.8 Ewe hoggets exhibiting oestrus 72–74 52 37.0 Ewe hoggets not exhibiting oestrus g 981 62.9 Ewes g 231 54.5 Ewe hoggets 60 843 59.1 Ewes 60 249 48.6 Ewe hoggets a Percentage of ewes lambing per inseminated. b Percentage of ewes pregnant per inseminated, determined by ultrasound 45–110 days after insemination. c Percentage of ewes pregnant at slaughter 50–100 days after insemination. d Semen frozen in pellets. e Semen frozen in 0.25 ml straws. g AI 12 h after oestrus detection. Ž . insemination after sponge removal 48, 60, 72 h was more critical in MAP- than in Ž . FGA-treated ewes Eppleston and Roberts, 1986 . Also, lambing depended on the w intravaginal device used for synchronisation of oestrus in the autumn 54.7 vs. 67.4 for Ž . x CIDR controlled internal drug release vs. Chronogest sponge , but not in the spring Ž . Ž . 59.2 vs. 56.2 respectively Wilson and Maxwell, 1989 . 3.6.5.2. Time of insemination. The appropriate time of insemination after sponge Ž . removal was found by different workers to be between 48 and 65 h. Early 36 h or late Ž . Ž 72–78 h insemination after sponge removal generally reduced the fertility see Table . 1 . Differences in the median time of ovulation in different breeds of ewe at different Ž . locations may explain the variations in fertility. Maxwell 1986a reported median times of 55.8 and 59.7 h after FGA sponge removal for teased and unteased Merino ewes respectively, compared with a mean time of 65.8 in the reports of Findlater et al. Ž . 1988a,b for Bluefaced Leicester = Swaledale ewes. In some reports where the time of ovulation was known, inseminations before ovulation were most effective, whereas in others later inseminations after the estimated time of ovulation were as efficient as inseminations before ovulation. Intrauterine insemination performed close to or coinci- dent with the time of ovulation resulted in low fertility. In superovulated ewes, the effective time of intrauterine insemination was observed to vary within the range of 44 to 64 h after removal of progestagen sponges. The differences may be attributed to variation in the precision and timing of ovulation due to Ž . different superovulation regimes. The use of gonadotrophin releasing hormone GnRH in superovulated ewes improved the synchrony of ovulation, and thus increased the fertility after intrauterine insemination. Most workers have not been able to demonstrate a beneficial effect of GnRH on the fertility of non-superovulated ewes. Superovulation was found to impair both transport and viability of spermatozoa after vaginal or cervical insemination, and also after late or early intrauterine insemination. Late insemination has been associated with abnormal egg fertilisation or failure of fertilised eggs to cleave. It has also been suggested that the uterotubal junction may act as a barrier to the transport of frozen–thawed spermatozoa in superovulated ewes after ovulation, and that laparoscopy may interfere with normal ova capture and transport by Ž the oviduct if performed close to ovulation in superovulated ewes reviewed by Salamon . and Maxwell, 1995b . 3.6.5.3. Dose of spermatozoa for insemination. Intrauterine insemination allows a decrease in the number of spermatozoa deposited in comparison with cervical insemina- tion. Nevertheless, account should be taken of the possible impairment of sperm transport through the uterotubal junction if the number of spermatozoa deposited in the Ž . uterus is excessively low Hunter et al., 1982 . Ž . Fertility data for ewes inseminated with a range 10 to 200 million total of frozen–thawed sperm doses are presented in Table 1. There are also reports on laparoscopic insemination with lower numbers of spermatozoa. Fertility was not differ- Ž . ent after deposition of 1 and 10 million total motile spermatozoa Walker et al., 1984 . The proportion of superovulated ewes with fertilised eggs after insemination of low Ž . numbers of spermatozoa 0.1, 0.5, 1.0 million total spermatozoa was better for tubal Table 2 Ž Results of AI with long-term frozen-stored semen tests 1–4 cervical, tests 5 and 6 intrauterine insemination . by laparoscopy a Ž . Ž . Test Period of storage years No. of ewes inseminated No. lambing or pregnant Ž . 1 3 172 91 52.9 Ž . 2 5 70 37 52.9 Ž . 3 7 143 74 51.7 Ž . 4 11 159 88 55.3 b Ž . 5 16 193 119 61.7 a Ž . 6 27 205 124 60.5 a At day 50. b Pooled data for 10, 20, 40 and 80=10 6 motile sperm doses. Ž . Ž 56, 70 and 100, respectively than uterine insemination 0, 11 and 40, . Ž . respectively Maxwell et al., 1993 . It seems that the minimum number of frozen–thawed motile spermatozoa which still yield satisfactory fertility after intrauterine insemination of ewes in synchronised oestrus is about 1 million. 3.6.5.4. Site of intrauterine insemination by laparoscopy. The site of intrauterine insemination may be a factor influencing fertility. Deposition of frozen–thawed semen in the middle and bottom of the uterine horns tended to be better than into the tip of the Ž . horns 53 and 53 vs. 44 lambing, respectively and there was also an advantage Ž . from insemination into both rather than only one horn Maxwell, 1986b . However, others have reported no difference between insemination in the tip or middle of the left Ž . or right uterine horn or at the junction of the horns Reinhold et al., 1990 , and no effect Ž . of site one or both horns of intrauterine insemination on fertility in synchronised Ž . superovulated Evans et al., 1984 or non-superovulated ewes comparing two horns vs. Ž . body of uterus Eppleston, 1993 . At present, intrauterine insemination by laparoscopy offers not only satisfactory and repeatable lambing results with frozen–thawed ram semen, but also allows a substantial reduction in the number of motile spermatozoa deposited. It should be noted, however, that a number of studies encouraged the belief that the technique of transcervical insemination has the potential to replace the laparoscopic method in some situations. Further research on its application and a better understanding of the requirements for Ž . cervical penetration is required. A recent study Maxwell et al., 1999 indicates that frozen–thawed spermatozoa supplemented with ram seminal plasma may yield accept- able fertility after cervical insemination. The efficiency and applicability of the methods needs confirmation.

4. Long-term frozen storage of semen