Abstract
There has been increased interest recently in the conservation of wild felids and preservation of valuable cat breeds. Assisted reproduction, by means of artificial insemination (AI), is an important tool for developing breeding programs for conservation. Optimal use of AI requires accurate data on semen conservation protocols and its long-term storage/survival. In this paper, semen cooling and freezing processes are described, with special emphasis on the results obtained in experiments performed in the domestic cat. Conception rates after AI in wild and domestic cats are also reported.
Introduction
There are 37 feline species and all of them, with the exception of the domestic cat, are threatened by extinction, as reported by the Convention of International Trade of Endangered Species (CITES 1973). Wild feline species quite often demonstrate poor breeding performance, both in captive and in natural conditions. One of the most important causes of infertility or sub-fertility is decreased genetic diversity, which is caused by inbreeding due to geographical isolation and population contraction (Wildt 1990). An improved reproductive performance in non-domestic Felidae is therefore needed and a successful breeding program frequently involves assisted reproduction techniques (ART), such as artificial insemination (AI).
The use of AI can reduce difficulties such as natural aggressiveness of these species, male–female behavioral incompatibility, or physical handicaps (Wildt 1990). Moreover, it reduces the risks of infectious disease transmission through copulation. With AI it is also possible to transfer semen from captive to wild populations orbetween wild populations geographically separated, which contributes to restoring genetic vigor. AI can be performed with fresh, cooled or cryopreserved semen, the latter providing long-term storage for postponed use.
In felids, the first attempts to prolong sperm viability and preserve its fertilizing ability were performed in the domestic cat. Its role is very important as a comparative model for studies aimed at non-domestic feline species, but ART can also be employed in the domestic cat to obtain kittens from valuable cat breeds.
The aim of this paper is a review of the results obtained in feline sperm conservation through cooling and freezing.
Semen conservation
For assisted breeding purposes, semen can be collected with an artificial vagina (Sojka and Jennings 1970, Sojka et al 1970, Zambelli andBelluzzi 1998), with an electroejaculator (Seager 1976, Platz and Seager 1978, Dooley et al 1983, Pineda et al 1983, Johnstone 1984, Herron et al 1986, Howard et al 1986) and from epididymides and vasa deferentia of isolated testicles (Howard et al 1986). Conservation protocols can be applied both to ejaculated and to epididymal spermatozoa. The latter has proven to be a precious source of sperm in case of orchiectomized or dead animals.
In order to preserve sperm quality, it is important to know physiological characteristics of the semen that are species-specific, as demonstrated by the poor results which were obtained in the past trying to apply to cat sperm the same diluents and procedures developed for cattle. Undiluted fresh cat semen, stored at +37°C, maintains motility in vitro for 60 min. This period can be prolonged up to 140 min if semen is stored at +23°C, and even more if diluted with an appropriate medium (Goodrowe et al 1989). Thus, great importance is attributed to diluent osmolality. The osmolality of cat semen ranges from 290 to 320 mOsm/kg and the ideal diluent should be about the same value. Hypotonic solutions cause swelling and membrane rupture, which are generally more detrimental than the shrinkage caused by hypertonic solutions (Glover and Watson 1985, Pukazhenthi et al 2002).
The presence of seminal plasma negatively affects spermatozoal fertilizing ability in the cat (McLaughling and Hamner 1974), therefore sperm centrifugation at low speed (300 g) and subsequent removal of seminal plasma significantly prolongs spermatozoal survival time. Better results can be achieved applying the swim-up process, which selects spermatozoa on the basis of motility and structural integrity (Howard et al 1990).
Semen cooling
Both ejaculated (Glover and Watson 1985, 1987, Pope et al 1989, 1991, Pukazhenthi et al 1999) and epididymal spermatozoa (Goodrowe and Hay 1993, Harris et al 2001) can be prepared for the cooling process. It is also possible to cool the testicles and then flush the epididymides and vasa deferentia in order to collect spermatozoa (Goodrowe and Hay 1993, Hay and Goodrowe 1993). The semen sample is extended with a diluent and subsequently cooled and stored at +5°C. This procedure allows a short-term storage: overnight (Goodrowe and Hay 1993), for 24 h (Glover and Watson 1985, Pope et al 1989, 1991), or sometimes even up to 5–7 days (Glover and Watson 1987, Harris et al 2001). Recently, Harris et al (2002) demonstrated that blastocyst development occurs after in vitro fertilization of cat oocytes with spermatozoa stored at +4°C for 14 days.
Some studies have focused on cooling diluents with the aim of finding the ideal medium to limit cold-induced damages on spermatozoal motility and morphology. Glover and Watson (1985) suggested a simple Tes-Tris (TEST) buffer solution of 292–325 mOsm/kg as adequate extender during sperm cooling. Egg yolk, the most effective agent to protect spermatozoa against cold shock, is commonly included in semen diluents, but it is not as effective for all species; and in fact it did not increase the survival of cat spermatozoa at +5°C (Glover and Watson 1987). The protective action of egg yolk is due to the lipoproteins included in the low-density fraction (LDF). This compound protects the cell surface by interacting with the membrane (Watson 1976) and experiments conducted with cat spermatozoa demonstrated that LDF is better tolerated than egg yolk in this species (Glover and Watson 1987). The inclusion of monosaccarides (glucose, fructose and galactose) appeared to be the easiest way to provide exogenous energy substrate, but none of them offered any real advantage during storage of cat semen (Glover and Watson 1987).
The cooling rate also influences spermatozoal survival. Pukazhenthi et al (1999) studied the sensitivity of felid spermatozoa to various rates of cooling temperatures and found that rapid cooling was more detrimental than slow cooling to cat spermatozoa morphological integrity. Even for epididymal spermatozoa, some authors reported a decreased motility in the semen cooled overnight compared to fresh semen (Goodrowe and Hay 1993). On the other hand, Harris et al (2001) comparing the effect of cooling on both ejaculated and epididymal spermatozoa after 5 days of storage, reported better results of motility in epididymal semen diluted in TEST-yolk buffer compared to ejaculated semen (69 vs 51.4%), with no significant difference in viability (91 vs 88.5%).
Recent work has confirmed that cat epididymal sperm cells survive after cooling, and their ability to interact with homologous oocytes is preserved. Interestingly, supplementation with antioxidants did not significantly influence motility and sperm morphology after storage (Leoni 1999), although lipid peroxidation of membranes by oxygen free radicals may be a cause of damage and loss of motility observed in cooled spermatozoa (Aitken 1995), and effects are also seen with frozen sperm (see subsequently).
Semen freezing
This procedure is particularly important for endangered species, as it is a valuable tool for creating effective semen banking. It has been successfully employed both in the domestic cat with ejaculated semen (Platz et al 1976, 1978, Pope et al 1991, Tsutsui et al 2000, Zambelli et al 2002) and epididymal semen (Hay and Goodrowe 1993, Lengwinat and Blottner 1994, Stachecki et al 1994), and in non-domestic felids with ejaculated semen (Howard et al 1986, Byers et al 1989, Donoghue et al 1992, Swanson et al 1996a, 1996b) and epididymal semen (Nelson et al 1999, Bartels et al 2000). Generally, semen can be stored in straws, ampules, or pellets (Howard et al 1986), although it has been shown for cat semen that the straws give better results than pellets (Pope et al 1991).
Prior to freezing, semen requires an equilibration period at +5°C for about 20 min. Then it is packaged in straws or ampules and exposed to liquid nitrogen vapor by suspending 4–5 cm above the liquid nitrogen, prior to the immersion and final storage. The temperature change may also be programmed with a controlled freezer, and a suitable freezing rate has been found to be −10°C/min from +5 to −80°C, before the immersion in the liquid nitrogen (Pope et al 1991). The pelleting technique involves pipetting single drops of semen into 3-mm diameter indentations made in a block of dry ice. After a 3-min interval, the block is inverted, plunging the pellets into liquid nitrogen.
The diluents for freezing cat spermatozoa are similar to those already mentioned for cooling, with a supplementation of cryoprotectant agents, with glycerol being the most widely used for this purpose. Glycerol is used at low concentration (4%) as cat spermatozoa seem to be especially sensitive to high concentration (Nelson et al 1999). It has also been reported that sperm motility in cat is more sensitive to changes in osmolality than membrane integrity, and removal of cryoprotectant in multiple steps with an isotonic solution minimizes loss of sperm motility and membrane disruption (Pukazhenthi et al 2002). However, an ideal freezing diluent for felid spermatozoa has not yet been defined, in fact sperm motility and acrosomal morphology are greatly affected by the freeze-thaw procedure. Cryopreservation of ejaculated semen was performed in the cat for the first time by Platz et al (1976) using a diluent consisting of 20% egg yolk, 11% lactose and 4% glycerol in de-ionized water. In 1992, Donoghue et al cryopreserved tiger semen in the samemedium and reported a decline in motility of 10–40%. Similar results were obtained by Byers et al (1989) using a TEST-yolk extender with 7.5% glycerol. Zambelli et al (2002) demonstrated that a freezing rate of 3.85°C/min for cat semen diluted in Tris with 20% egg yolk and 4% glycerol, gave better results in spermatozoal motility and morphology preservation than faster freezing rates. Using epididymal semen, Hay and Goodrowe (1993) compared three different extenders containing 20% egg yolk and 3% glycerol: TE (Tris buffer, citric acid and fructose), TC (Tris buffer, citric acid and glucose) and CP (lactose). In their study progressive motility of spermatozoa was reduced approximately by 20% from prefreeze values, in presence of Tris buffer and monosaccharides in the diluent TE and TC.
Motility stimulants such as caffeine, pentoxifylline and 2′-deoxyadenosine had a dose-dependent effect on motility and produced a hyperactivated motion of thawed cat epididymal spermatozoa reducing the decrease in motility by approximately 10% compared to the prefreeze value (Stachecki et al 1994).
As already mentioned, lipid peroxidation can induce direct membrane damage or changes in membrane structure or fluidity causing rapid and irreversible loss of motility. This event occurs in spermatozoa after cooling and even more after freezing–thawing processes.
In order to face the accumulation of reactive oxygen species during storage, the effect of adding the antioxidant taurine (25 or 50 mM) to the diluent used for freezing epididymal cat semen has been evaluated (Luvoni et al 2002). Taurine, a sulfur-containing β-amino acid, is present in the reproductive tract of several mammals and recently high concentrations have been found in spermatozoa, seminal plasma and epididymal fluid of domestic cat (Buff et al 2001). The results indicated that after thawing, sperm motility was better preserved in the presence of taurine (25 mM: 52.2±5.9%; 50 mM: 53±3.7%) compared to in its absence (23.8±4.6%), with no differences between the two concentrations of taurine added.
Methods of thawing frozen semen vary widely. Straws are thawed rapidly in a +35°C water bath, ampules are allowed to thaw slowly in an ice–water bath for 10 min before semen is transferred into the insemination catheter. Pellets are thawed rapidly by plunging them into a 0.9% saline solution warmed to +37°C (Howard et al 1986).
Conception rates after AI
In spite of the reduced spermatozoal motility and morphology after thawing, frozen semen has been employed for AI purposes and the results are compared with fresh semen AI in Table 1.
Conception rate in domestic cat inseminated with fresh and frozen semen
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The first kitten born following intravaginal insemination with frozen semen was obtained by Platz et al (1976). Two years later, Platz et al (1978) reported a pregnancy rate of 10% after intravaginal insemination. Such a poor result could be explained both as a consequence of the site of insemination (vagina and not uterus) and as a result of an inaccurate timing of the insemination with the expected onset of ovulation. It is well known that the site of semen deposition greatly affects the success of AI with frozen semen, because the survival time of thawed spermatozoa is significantly reduced. The development of surgical or laparoscopical intrauterine insemination, that allows sperm to be deposited close to the site of fertilization, improved the results in pregnancy rates to 57% (Tsutsui et al 2000).
The application of frozen semen AI to non-domestic cats remains a problem. Attempts at intravaginal insemination of anesthetized non-domestic cats have rarely been successful (Dresser et al 1982, Howard 1999), primarily due to compromised sperm transport and uncertainty about the ovulatory status of gonadotropin-treated females. However, laparoscopic AI has been achieved in seven non-domestic felid species. In three of them (cheetah, leopard cat and ocelot), offspring have also been produced after AI with both freshly collected and frozen-thawed spermatozoa (Howard and Doherty 1991, Swanson et al 1996a, Howard et al 1997, Howard 1999).
The development of a technique for non-surgical intrauterine insemination, that might ensure satisfactory pregnancy rates, is desirable. A practical contribution to this topic came recently from Zambelli and Castagnetti (2001). They used a tomcat catheter with a 1 mm diameter and a nob-pointed needle inserted at the cut end to inseminate eight queens with frozen semen. Transcervical catheterization was possible in only five animals, but in all of them, embryos were recovered after ovarioisterectomy performed 5 days later.
Conclusions
One important way to prevent extinction in non-domestic felids is to establish a ‘frozen semen bank’ and to apply ART by means of AI. This requires developing conservation protocols for semen, in order to improve reproduction that in these animals is quite poor, because of geographical isolation and population contraction that increase inbreeding and reduce genetic diversity. Moreover, the difficulties concerned with handling these animals, which make it impossible to monitor and to manipulate the female estrous cycle, contribute to the need of spreading ART. In the domestic cat AI is indicated to optimize reproductive performances in valuable animals or when cat is studied as a comparative model for feline endangered species. Feline semen conservation through cooling or freezing has so far brought some encouraging results, such as the birth of offspring after AI with stored semen, but an optimal procedure is far from being defined. There is a need for further investigations focused on the diluents (such as the supplementation with cell protectants), combined with the rate of cooling to freezing temperatures to preserve integrity and fertilizing potential of spermatozoa.