Ž .
Animal Reproduction Science 62 2000 77–111 www.elsevier.comrlocateranireprosci
Storage of ram semen
S. Salamon, W.M.C. Maxwell
Department of Animal Science, UniÕersity of Sydney, Sydney, NSW 2006, Australia
Abstract
Storage of ram semen in liquid and frozen state, the diluents used for both methods, processing, cooling, freezing and thawing of semen are reviewed. Factors influencing the fertility
of stored semen and methods used for improvement are discussed, and fertility results of long-term frozen stored ram semen are also given. q 2000 Elsevier Science B.V. All rights
reserved.
Keywords: Spermatozoa; Liquid and frozen storage; Diluents; Protective agent; Freezing; Thawing; Cervical and intrauterine insemination
1. Introduction
Ž .
Investigations on artificial insemination AI of sheep were initiated at the beginning Ž
. of the twentieth century by Ivanov 1907, 1912 , whose studies on diluting media and
reproduction led to the development and the practical application of AI with farm animals. After the First World War, further intensive studies, under the leadership of
Milovanov, were undertaken in the former Soviet Union, and by the early 1930s AI with fresh and diluted semen was used on a large scale in sheep breeding programmes.
The need to fertilise large numbers of ewes with semen of outstanding rams required transport of semen from the points or centres of collection to the sites of insemination at
more distant farms. The necessity to use the rams over extended periods, or at different times of the year, stimulated research on storage of spermatozoa under artificial
conditions.
This could be achieved by methods that reduced or arrested the metabolism of spermatozoa and thereby prolonged their fertile life. Accordingly, investigators exam-
Ž . Ž
. ined storage of semen in i a liquid unfrozen state, using reduced temperatures or
Corresponding author. Tel.: q61-29-351-4864; fax: q61-29-351-3957. Ž
. E-mail address: chismvetsci.usyd.edu.au W.M.C. Maxwell .
0378-4320r00r - see front matter q 2000 Elsevier Science B.V. All rights reserved. Ž
. PII: S 0 3 7 8 - 4 3 2 0 0 0 0 0 1 5 5 - X
Ž . other means to depress sperm metabolism; and ii in a frozen state which involved
preservation at sub-zero temperatures. Comprehensive reviews have been published on Ž
. Ž
liquid Maxwell and Salamon, 1993 and frozen storage of ram semen Salamon and .
Maxwell, 1995a,b .
2. Liquid storage of semen
Ž The main methods of storage of semen in a liquid state are storage at reduced 0–5 or
. 10–158C and at ambient temperatures by reversible inactivation of spermatozoa.
2.1. Storage at reduced temperatures Ž
. Milovanov 1940, 1951 described the early work and discussed the problems of
Ž storage of ram spermatozoa in a chilled state. Whereas British investigators Chang and
. Walton, 1940 and some Soviet workers claimed that 10–158C was the ‘‘optimum’’
Ž .
temperature for liquid storage, most Soviet investigators reported that survival of ram and bull spermatozoa was better after storage at 0–58C.
When semen was stored at a low temperature, care had to be taken not to subject the spermatozoa to cold shock. This caused irreversible changes in sperm cells cooled to
temperatures close to 08C. The detrimental effects of cold shock, discovered in 1931 Ž
. Milovanov, 1951 , were overcome either by gradual cooling of semen from room
temperature, or by addition of lipid to the diluent. The value of lipids from various Ž
. sources egg yolk, testicles, corpus luteum, brain and soy beans in the protection of
spermatozoa against cold shock was initially reported by Milovanov and Selivanova in Ž
. Ž
. 1932 cited by Milovanov, 1962 . Lardy and Phillips 1939 and Phillips and Lardy
Ž .
1940 applied these observations in the development of the egg yolk–phosphate diluent for the preservation of bull semen. Another generally accepted storage medium, the egg
Ž .
yolk–citrate diluent, was elaborated later by Salisbury et al. 1941 and Willett and Ž
. Salisbury 1942 .
Before 1940, the main diluent used in the former Soviet Union for liquid storage of Ž
. Ž
. ram semen was glucose–phosphate–phospholipid soy extract medium GFO-5 . This
was subsequently replaced by glucose–citrate–egg yolk as the generally accepted Ž
. Ž
diluent for ram semen Milovanov, 1962 . The egg yolk component and more likely its .
high molecular weight low density lipoprotein fraction , apart from providing protection against cold shock, has been shown to reduce the loss of acrosomal enzymes and prevent
degenerative changes in the acrosome during liquid storage. The composition of the citrate diluent as used nowadays is:
Ž .
2.37 g sodium citrate 2H O
2
0.50 g glucose 15 ml egg yolk
100,000 i.u. penicillin 100 mg streptomycin
glass distilled water to 100 ml
Although fructose is the only simple carbohydrate present in ram semen, spermatozoa can also metabolise glucose and mannose when these sugars are included in the storage
diluent. No other sugars act as energy sources, but a range of sugars have been examined for their ability to preserve the motility of spermatozoa. Most investigators
Ž used glucose and fructose as sugar components of the diluent. Some other sugars e.g.
. sucrose and lactose may only act extracellularly to maintain the osmotic pressure of the
diluent and membrane integrity of the sperm cells during storage. Attempts to replace the traditional citrate buffer with glycine or addition of glycine to
the citrate buffer gave contradictory results regarding the survival of spermatozoa, and were abandoned. Other diluents investigated, such as citrate–glycol–egg yolk–mucinase
Ž .
Ž .
Sokolovskaja et al., 1956 and citrate–boric acid–egg yolk Yoshioka et al., 1951 , were variants of the above citrate diluent and remained only in experimental use.
During the early and mid-1970s, several studies concentrated on the use of organic buffers, on the basis that these have better buffering capacity than phosphate or citrate,
are relatively non-toxic to living cells, and may penetrate the sperm cell thereby acting as intracellular buffers against pH shifts, or may increase the tolerance of the cell to an
intracellular increase in monovalent cations. Organic buffers often increase the overall tonicity of the diluent, which is important when the semen is stored.
Ž .
Ž .
Diluents containing tris hydroxymethyl aminomethane Tris as the main component Ž
have been examined for storage of semen from the bull, boar and ram reviewed by .
Visser, 1975 . Tris concentrations ranging from 10 to 50 mM were reported to have little or no effect on motility and metabolism of ram spermatozoa. Subsequently, higher
concentrations of tris were reported to be advantageous in diluents for chilled storage. More research is required, particularly on those buffers with efficient hydrogen ion
buffering capacity in the ‘‘sperm tolerant’’ range of pH 6.5–7.5, for example Tes w
Ž .
x w
N-Tris hydroxymethyl -methylaminoethane-sulfonic acid , Hepes N-2-hydroxyethyl-
X
x w Ž
. x
piperazine-N -2-ethanesulfonic acid , Mops 3- N-morpholino propane sulfonic acid , w Ž
. x
Ž . Ž
Mes 2 N-morpholino ethane sulfonic acid and Pipes piperazine-N, N-bis 2-ethane
. sulphonic acid .
Ž Nowadays, the tris-based medium is a recommended extender for ram semen Evans
. and Maxwell, 1987 , and its composition is as follows:
3.63 g tris 0.50 g fructose
1.99 g citric acid 14 ml egg yolk
100,000 i.u. penicillin 100 mg streptomycin
glass-distilled water to 100 ml
Whole, skim or reconstituted milk have also been used for many years as extenders for ram semen. The success of this diluent has been attributed to its protein fraction,
which may act as a buffer against changes in pH and as a chelating agent against any heavy metals present. It may also partially protect spermatozoa during reduction of
temperature for storage. Cows’ milk has been preferable to the milk of other species.
Before dilution of semen, the whole, skim or reconstituted milk should be heated at 92–958C for 8–10 min to inactivate the lactenin in the protein fraction which is toxic to
spermatozoa. The skim milk powder must be carefully selected because some samples may not be satisfactory for preparation of diluent. The reconstituted milk diluent is
prepared by dissolving 9 g of skim milk powder in 100 ml of glass-distilled water. To
Ž control microbial growth, antibiotics
e.g. 1000 i.u. sodium penicillin and 1 mg .
streptomycin sulphate should be added to each milliliter of whole, skim and reconsti- tuted milk diluents.
Some investigators found that skim milk was better than whole milk for storage of semen at 2–58C. When combined with antibiotics, it was as effective as egg yolk–glu-
cose–citrate diluent for chilled storage of ram semen. Addition of 5 egg yolk and 1 glucose to the skim milk was claimed to improve the viability of spermatozoa during
Ž .
chilled storage Feredean et al., 1967 . Regarding reconstituted skim milk, French workers found that viability and fertility of ram spermatozoa was better after storage for
Ž .
8–16 h at 158C than at 58C Colas et al., 1968, 1974 . Ž
. In past years, commercially available ultra-heat-treated UHT ‘‘long-life’’ milk has
proved a satisfactory diluent for fresh semen and also for maintaining the viability of spermatozoa during liquid storage. The UHT milk is sterile, does not require heating and
may be used directly as a diluent without further treatment; a freshly opened carton of UHT milk is required for use each day.
Ž .
Recent studies on the suitability of a ‘‘chemically defined’’ diluent RSD-1 , with and without combination with antioxidants, for liquid storage of ram semen have yet to
Ž .
improve fertility over other diluents Upreti et al., 1997 . 2.2. Storage at ambient temperatures by reÕersible inactiÕation of spermatozoa
The first experiments on the inhibitory effect of carbon dioxide on the motility of spermatozoa of different species were performed in 1924 by Krshyshkovsky and Pavlov
Ž .
cited by Milovanov, 1962 . More than three decades later, this method was re-examined Ž
. and a new extender for bull semen, called the Illini Variable Temperature IVT diluent,
Ž .
was elaborated Salisbury and VanDemark, 1961 . The diluent required gassing with carbon dioxide before use.
Another diluent, also developed for storage of bull semen at ambient temperature, the Ž
. Cornell University Extender CUE , is a self-gassing medium in which the carbon
Ž dioxide is derived from the reaction of citric acid and sodium bicarbonate Foote et al.,
. 1958 . Both diluents have been modified for use with ram semen; they were saturated
with carbon dioxide by gassing to pH 6.3 for about 10 min before use, or were ‘‘self-carbonating’’ as a result of their carbonate or bicarbonate and acid content.
Ž .
As an alternative to gassing, diluents containing volatile organic oxalic and acetic Ž
. acids were also examined Habibulin, 1963 .
2.3. Fertility of semen after liquid storage and factors influencing it There are many reports on the fertility of liquid stored semen after cervical insemina-
tion, mainly from the former Soviet Union. A critical assessment of most reports is
difficult, as they do not provide sufficient information on essential experimental procedures. Details such as type or composition of diluent, temperature of storage,
dilution rate, dose of inseminate, type of teaser rams used for the detection of oestrous Ž
. ewes vasectomised or aproned entire rams , number of inseminations within one oestrus
Ž .
and the method of determining the fertility lambing data were often not stated. Several papers were in the form of reports of selected data from large-scale field insemination
programmes, rather than from proper experiments. Ž
. In critical studies reviewed by Maxwell and Salamon, 1993 , fertility declined
rapidly when semen stored for more than 24 h was used for cervical insemination. The decrease in fertility was at a rate of 10–35 per day of storage. Thus, while 68–75 of
ewes lambed from insemination with fresh semen in a single cycle, the lambing rates for semen stored for 24, 48 and 72 h were 45–50, 25–30 and 15–20, respectively.
Irrespective of the diluent, dilution rate, temperature or conditions of storage, the spermatozoa deteriorated as the duration of storage increased. The main changes that
occurred during storage included reduction in motility and morphological integrity of spermatozoa. These changes may be contributed to by the accumulation of the toxic
Ž .
products of metabolism, mainly of reactive oxygen species ROS formed through lipid peroxidation of the membranes of spermatozoa. The above events are accompanied by a
decline in transport and survival of spermatozoa in the female reproductive tract and reduction in fertility.
The cervix constitutes the initial barrier to the ascent of spermatozoa. By comparison to fresh spermatozoa, a relatively small proportion of the stored cells deposited at the
external os cervix during insemination penetrate the cervical canal and migrate through the tract of the ewe to the site of fertilisation. Establishment of an adequate cervical
sperm population is important with the insemination of sheep, as spermatozoa may continuously ascend to the oviducts from this reservoir. However, spermatozoa function-
ally affected during liquid storage may not migrate, or may migrate slowly, and their survival in the female tract is also reduced to about half that of fresh spermatozoa
Ž
. Lopyrin, 1971 . Attempts to improve the transport of spermatozoa from the posterior
Ž .
cervix to the oviducts of oestrous ewes by prostaglandins PGF a or PGE added to
2 2
Ž the stored semen have given conflicting results reviewed by Maxwell and Salamon,
. 1993 .
Ž .
It is also possible that the process of liquid storage like that of frozen storage advances the maturation of sperm membranes, thus increasing the proportion of capaci-
tated and acrosome reacted cells. Capacitated spermatozoa have reduced viability and limited fertile life; or they may be rendered incapable of fertilisation if they are further
Ž aged in the female reproductive tract after cervical insemination Maxwell and Watson,
. 1996 .
Early embryonic mortality is considered to be a further, or even one of the main causes of low fertility. The condition of gametes at the time of fertilisation is known to
affect embryonic survival. An increase in abnormalities of embryonic development associated with ageing of spermatozoa has been observed for several species, and
considerable attention has been focused on possible changes in the haploid genome of
Ž .
the sperm cell due to ageing Salisbury et al., 1976 . There is some evidence that embryonic loss may be increased when stored spermatozoa are further aged in the
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
