Perspective Chapter: Wild and Domestic Cats Semen Cryopreservation – Main Aspects and Perspectives

Abstract

Wild and domestic cats can benefit effectively from the use of cryopreserved semen. Different breeding techniques have been applied using cryopreserved semen to produce offspring in wild and domestic cats. To date, the success of these techniques in cats is still inadequate. The majority of sperm tend to lose motility and viability following thawing. Semen must be handled carefully, cryopreserved, and thawed by improving current protocols in order to produce post-thaw sperm of acceptable quality and to result in conception after insemination. The quality of sperm obtained following semen cryopreservation process might be beneficial to the success of reproduction performance. To enhance post-thaw recovery of sperm and fertility, it is crucial to have a thorough understanding of the sperm physiology of the species. These factors include the right diluent, sperm dilution, cooling, and thawing rate. Cryopreservation of semen across cat species often results in poor post-thawed sperm quality, including a decrease in motility and acrosomal integrity of up to 30–50% and a rapid decrease in progressive motility within hours of thawing. Due to the low number of sperm and compromised post-thawed sperm quality, laparoscopic-assisted reproduction is frequently used for artificial insemination procedures in Felids.

1. Introduction

Most of the global Feline populations are in decline in their natural habitats, with roughly half of them categorized as threatened according to the International Union for Conservation of Nature (IUCN) red list of threatened species (IUCN, 2021). Out of 36 known species of Felines, domestic cats (Felis catus) are the only ones not facing the risk of extinction [1]. However, wild cats are classified as threatened, vulnerable, or endangered due to poaching and habitat destruction. Domestic cats (Felis catus) are largely kept as pets and breeding has not been organized on a large scale as with other domestic animals. In the Feline species, the exchange of breeding animals takes place in laboratory animal research facilities, yet commercial activities concerning the trading with cryopreserved semen are rare. In order to secure the continued existence of populations, especially of those highly endangered species, numerous in situ and ex situ conservation initiatives are being implemented globally. Cryobanking is essential in reproductive facilities to preserve endangered species’ gametes for future use [2].

Breeding of endangered species in zoological gardens or specialized conservation centers is an ex situ conservation method, though it is not always successful. Issues like intraspecies aggression, incompatibility, inadequate sexual behavior, lack of libido, and incompetent mating [3] can present a challenge when replacing “ineffective” individuals or breeding pairs due to the limited number of animals available in captivity and difficulties with exchanging animals between institutions. Assisted Reproductive Technologies (ARTs) are crucial for the success of conservation programs aiming to raise the population size and genetic diversity of wild cats [4]. The ARTs are increasingly being recognized as an essential element of captive breeding programs for certain wild cat species. To counteract this, ARTs such as cryopreservation of gametes from the lifetime or postmortem, as well as the use of Artificial insemination (AI), In vitro fertilization (IVF), and embryo transfer could be employed to maintain genetic diversity. Maintaining a stable and healthy population of cats in captivity is difficult due to their reduced reproductive success compared to the wild; however, it is a critical measure to help protect threatened cat species [5]. Although AI in cats is more challenging than in other species due to difficulty in semen collection and oestrous cycle management [6], demand for semen collection, cryopreservation, and subsequent AI is on the rise for the purpose of preserving valuable genetic material [7] and controlling diseases such as respiratory infections, or circumventing psychological or physical constraints on breeding. Cryopreserved semen for AI can be an alternative to importing founder animals from foreign countries, which can potentially improve global management programs [8] and promote genetic diversity between zoo and wild Felids [9].

Cryopreservation is a useful technique for conservation efforts, as it allows for the preservation of cells and tissues of wild Felids over a long term using subzero temperatures [10]. Cryopreservation of semen offers the advantage of transferring genetic material between distant locations, rather than just living animals, guaranteeing that genetic diversity is not lost if the animals die without reproducing [11]. Cryopreservation of semen from domestic cats has been successfully achieved with straw freezing in a medium containing glycerol and egg yolk, as well as an alternative medium replacing the egg yolk with soy lecithin. Additionally, ultra-rapid freezing of semen directly into liquid nitrogen (LN₂) has demonstrated promising results. Nevertheless, these methods yield fewer positive outcomes when compared to cryopreservation methods and media developed for other domesticated mammals [12, 13]. Cryobanking appears to be a crucial tool in maintaining biodiversity; storing material serves as a “safety net” in case other conservation efforts fail. Furthermore, the exchange of cryopreserved material is more convenient than that of live animals in zoological gardens. Long-term storage of cats’ semen, which is mostly performed by slow freezing method, would allow the establishment of effective semen banking. Unfortunately, due to the very small volume of the ejaculate with a low sperm count and the poor semen quality of the Felids, poor results are obtained when cryopreserving cat semen [14].

Literature reported that the majority of 37 species in the family of Felidae are uncommon and endangered. This includes wild Felids, and domestic cats, which have been used as experimental models in reproduction research [15]. Wild and domestic cats are important research models for figuring out the biological processes governing reproductive success in Felidae species [16]. Our contemporary reality, which has significant consequences for wildlife conservation and threatens the planet’s biodiversity, is growing exponentially, driven by human population expansion and resource use [17]. The genetic management of small populations in breeding facilities, zoos, and parks could potentially incorporate assisted reproductive methods linked to genome resource banking [18]. In breeding projects like IVF for fertility assessment, genome banking, and other assisted reproductive techniques, gathering high-quality semen from the zoo and wild animals is crucial [19]. An essential technique for the preservation of wild and domestic cat populations is the application of reproductive biotechnologies [20]. The aim of this chapter was to provide an overview of the cryopreservation of semen from wild and domestic cats.

2. Semen collection methods in wild and domestic cats

Techniques have been established to preserve semen in both domesticated and wild Felids. Collecting semen from domestic cats and many small wild Felids typically yields a limited amount of semen, with only 10–50 million sperm being obtained per ejaculate. Male cats usually produce an ejaculate with a relatively small volume and a low number of sperm [21]. The main purposes for semen collection are to store it for future use (cryo-semen banking), recover sperm after death (postmortem sperm rescue), use it for AI, IVF, and to assess fertility. It is essential to choose the better methods of semen collection to maximize any male’s reproductive capabilities. There are a few commonly used approaches for cat species, such as the artificial vagina, electro-ejaculation, collection from the epididymis, and a newly developed method of urethral catheterization.

2.1 Artificial vagina

Using an artificial vagina is a common technique employed in catteries and research colonies, where semen needs to be gathered regularly for AI, semen preservation, or other reasons from cats that are trained for the task. The artificial vagina is made up of a latex bulb from a medicine dropper and a plastic 1.5 mL microtube (Eppendorf) [22]. The top of the artificial vagina, which is securely fastened with rubber bands, is created by cutting the bulb from a medicine dropper. These can be purchased commercially from various manufacturers or homemade, but they must be watertight because any direct contact with water will kill the sperm. Once the artificial vagina is securely fastened, the male can be prepared for semen collection. This typically involves cleaning the genital area and allowing the male to mount the female, and introducing the penis into the artificial vagina before the male inserts it into the natural vagina when the penis is fully erect. Ejaculation can be stimulated with gentle movements [22]. This approach offers several advantages, including a low cost of the equipment and no need for restraining the cat. Furthermore, one technician can perform the collection. It is known that artificial vagina semen collection can be more successfully utilized in domestic cats than in wild cat species. Domestic cats are more accustomed to human interaction and handling, making the collection process less stressful for both the cat and the collector. However, training the wild cats to utilize an artificial vagina prior to collection is possible, whereby the male is allowed to approach the female as it would in the wild, to promote heightened sexual arousal. Sojka et al. [23] first described the use of an artificial vagina to collect semen from tom cats, which was then used to perform the first AI in cats. Tanaka et al. [24] reported that the artificial vagina can be used for up to 10 days consecutively, twice a day, without deteriorating the sperm quality. Many articles discussing Feline semen collection using an artificial vagina subsequently focused on examining semen quality or the application of semen samples obtained via this method in AI and other assisted reproduction methods [25].

2.2 Electro-ejaculation

Electro-ejaculation is an effective method of semen collection in cats, which may be applicable to other animals if they can be safely anesthetized [24]. It has certain advantages over the use of an artificial vagina in the context of an untrained or aggressive male, or in the absence of a teaser queen [26]. Studies have reported that electro-ejaculation is safe, with no significant histological or endoscopic lesions in the rectum following the procedure [27]. The main drawbacks are the need for anesthesia and the cost of the necessary equipment. Furthermore, the particular anesthetic protocol employed can have an impact on both semen quality and the rate of success [28]. The most frequently used anesthetic for cats undergoing electro-ejaculation is ketamine, which may be given alone (20 mg/kg intramuscularly) or in combination with medetomidine (5 mg/kg intramuscularly) and 80 g/kg intramuscularly, respectively [29]. Additionally, a successful semen collection by electro-ejaculation has been reported using an anesthetic protocol that includes 30–40 g/kg intramuscularly of dexmedetomidine along with 3–5 mg/kg intramuscularly of ketamine [7].

2.3 Epididymal recovery

Epididymal sperm remains mysterious. Described as immature, immotile, and infertile, the epididymis is the organ where sperm reach maturity, develop motility, and become fully capable of reproduction [30]. As such, epididymal semen can be utilized to preserve the genetics of prized breeding cats, particularly those that are unable to ejaculate, require castration for medical care or unexpected death occurs. When collecting epididymal semen, certain elements may have a harmful influence on the quality and freezability of the semen. As a result, removing these elements can increase the freezability of epididymal semen and consequently improve post-thaw semen quality and the success of assisted reproductive technologies. Collection of epididymal sperm can be done by flushing with a 29-gauge needle [31, 32], compressing with anatomical forceps [33, 34], slicing with a scalpel blade or scissors [35, 36, 37], or puncturing with a 30-gauge needle [38]. Having a complete understanding of the role of the epididymis as well as the specific features of sperm obtained from the epididymis compared to those from ejaculation is essential for the collection and cryopreservation of epididymal semen.

2.4 Urethral catheterization

Ejaculation has been induced in domestic and laboratory animals with Alpha 2 (α₂)-adrenergic agonists [39]. Zambelli et al. [40] developed a method for collecting semen in cats called association with urethral catheterization with α₂-adrenergic agonists. The technique of urethral catheterization for semen collection, which has been employed over the last couple of decades [40], has advantages over the artificial vagina and electro-ejaculation techniques in that it does not require any special equipment or a trained animal, and can be used in any country without restrictions. The urethral sperm collection technique involves introducing a 1.3 mm Feline catheter through the penile urethra after sedation and administration of an α₂-adrenergic agonist, which stimulates the adrenoreceptors in the deferent duct and releases semen into the penile urethra [41]. Medetomidine and dexmedetomidine are two α₂-adrenergic agonists used to induce ejaculation in domestic cats. Medetomidine aids in drowsiness, analgesia, and muscular relaxation [40], while dexmedetomidine is the most recent and selective agonist with effects such as drowsiness, analgesia, and anxiolytics [42]. Studies have shown that the two α₂-adrenergic agonists are effective at inducing ejaculation in domestic cats, which is important for assisted reproductive processes in wild cats. The results of sperm collection following the administration of high doses of dexmedetomidine (60 g/kg) or medetomidine (120 g/kg) and low doses of the two drugs (50 and 25 g/kg, respectively) have been compared [43]. Although the semen collected by the urethral catheterization method may have some macroscopic and microscopic distinctions compared to other techniques, it is still suitable for cryopreservation [44] and can be utilized for IVF [44, 45] or AI, similar to sperm collected using an artificial vagina or electro-ejaculation. The use of this method is on the rise for research on both domesticated and wild cats [44, 46] due to its similarity in producing high-quality semen like other methods (electro-ejaculation), in addition to its cost efficiency and reduced contamination of the semen sample [47]. This approach is highly valuable in clinical settings, as it is a cost-effective and straightforward way to produce an ejaculate. Samples of semen obtained through urethral catheterization tend to have a lower overall quantity, but a higher concentration of sperm (10⁶/mL) compared to those obtained through electro-ejaculation. Nonetheless, the total number of sperm per ejaculation is not significantly different between the two methods.

3. Semen macroscopic and sperm microscopic analysis in wild and domestic cats

In terms of knowing their reproductive physiology, Felidae species have often been underrepresented [48]. Using quantitative sperm functional techniques for sperm cryopreservation testing and functionally high-quality sperm insemination for the artificial propagation of cat species is still challenging. Given that, genetic donation, it is possible to exert stronger selective pressure on males (tom) than on females (dam/queen), male contribution to Feline reproductive efficiency is of utmost importance [15]. Males should therefore be thoroughly inspected and assessed to enable better selection. This includes evaluating semen samples, in the right way to ascertain their microscopic properties, such as motility, vigor, and sperm morphology, as well as their macroscopic qualities, such as volume, color, odor, and pH [49]. Clinically, the existence of motile, morphologically normal sperm in infertile individuals shows that they may be fertile [50]. New techniques for quantitatively evaluating sperm quality include advanced microscopy (image analysis) techniques like computer-aided sperm analysis (CASA) and sperm flagellar analysis [48].

Various characteristics of semen have been observed in several Felid species (Table 1). Although semen sample volumes may vary depending on the size of the species, the sperm concentration (which can be as low as a few hundred million sperm per milliliter) and sperm morphology remain uniform among Felids.

Felid species	Semen volume (mL)	Sperm concentration (million sperm/mL)	Sperm characteristic	Semen collection method	References
Leopard (Panthera pardus nimr)	1–5	50–100	Teratospermia	Electro- ejaculator	[51]
Lion (Panthera leo)	1–10	10–65	Teratospermia in some individuals	Urethral catheterization, Electro-ejaculator	[52]
Jaguar (Panthera onca)	2–9	5–50	No teratospermia	Urethral catheterization, electro-ejaculator	[52]
Puma (Puma concolor)	1.5–3.5	10–23	Teratospermia	Urethral catheterization, electro-ejaculator	[52]
Snow leopard (Panthera uncia)	1–2.5	10–40	Close to teratospermia	Urethral catheterization, electro-ejaculator	[52]
Clouded leopard (Neofelis nebulosa)	0.5–1.5	25–200	Teratospemia	Electro-ejaculator	[52]
Domestic cat (Felis catus)	0.1–0.2	100–500	Some individuals with teratospermia	Urethral catheterization, electro-ejaculator	[52]
Iberian lynx (Lynx pardinus)	0.3–0.7	5–25	Teratospermia	Urethral catheterization, electro-ejaculator	[52]
Flat-headed cat (Prionailurus planiceps)	0.1	50–60	No teratospermia	Electro-ejaculator	[53]
Jungle cat (Felis chaus)	0.07	70–80	No teratospermia	Urethral catheterization	[19]
Black-footed cat (Felis nigripes)	0.25	130–150	No teratospermia	Electro-ejaculator	[27]
Sand cat (Felis margarita)	0.2	200–250	Teratospermia	Electro-ejaculator	[27]

Table 1.

The main characteristics of semen and sperm in representative Felid species are reported here.

3.1 Semen volume

The precise volume of the ejaculate is measured after collection; it is ideal to use a pipette and measure the volume in microliters [54]. Cats generate a little amount of semen every ejaculation, often between 0.01 and 0.7 mL [39, 55]. According to Rijsselaere and Van Soom [56], the relatively small volume of the ejaculate, which sharply reduces the number of sperm assessments, is one of the most significant drawbacks for sperm evaluation in the Feline species.

3.2 Sperm concentration

According to previous reports, the cat ejaculate typically contains 8 to 30 million sperm, however, this number can range from 3 to 153 million sperm [39, 57]. A hemocytometer is used to measure the concentration of sperm at a 1:100 dilution in Feline species [54]. Studies have revealed that at least 80 million sperm must be deposited within the vagina for this procedure to achieve around 80% conception rate [24].

3.3 Sperm motility

Sperm should be progressively motile in order to reach and fertilize the egg/oocyte; for this reason, sperm motility is a crucial factor during both natural and aided conception [50]. It is well established that sperm motility is strongly correlated to IVF success and pregnancy outcome [58]. Sperm motility depends on the form and function of the flagella, and numerous reports link low motility to genetic flaws [59]. Immotile sperm can be present in the ejaculate due to testicular and/or epididymal dysfunction [50].

Sperm motility is normally measured using CASA calibrated for Feline sperm, low-power microscopy on a warmed microscope slide, or by estimation [54]. The CASA system is able to examine the spermatic parameters (%) such as total motility and progressive motility, fast, medium, slow, and static sperm subpopulations, Average Path Velocity (VAP m/μs), Curvilinear Velocity (VCL m/μs), Straight-Line Velocity (VSL m/μs), Linearity Index (LIN %), Straightness Index (STR %), Amplitude of Lateral Head Displacement (ALH μm), and Beat Cross Frequency (BCF Hz) [18]. The CASA has proven to be a valuable diagnostic tool for infertility clinics, providing objective analysis of sperm motility as well as establishing quality control standards for consistent semen analysis [60]. The CASA settings vary among each species, however, Felines (domestic or wild breed) are the same settings (Table 2).

Parameter	Setting
Frame rate (frames/second)	60
Duration of data capture (frames)	40
Minimum path length (frames)	40
Minimum motile speed (μm/second)	10
Maximum burst speed (μm/second)	1200
Distance scale factor (μm/pixel)	0.9348
ALH path smoothing factor (frames)	7
Cent. X search neighborhood (pixels)	4
Cent. Y search neighborhood (pixels)	2
Cent. Cell size minimum (pixels)	2
Cent. Cell size maximum (pixels)	9
Path. Max. interpolation (frames)	2
Path prediction percentage	10

Table 2.

Parameter settings for tracking Felid sperm using the CASA system [60].

ALH: Amplitude of lateral head displacement.

3.4 Sperm morphology

Domestic cats frequently exhibit poor sperm morphology (teratozoospermia) and decreased semen quality out of season, although the precise causes and mechanisms remain unknown [61]. Cat sperm typically have a lengthy tail, prominent midpiece, and oval head [54]. It has been asserted that domestic cats frequently produce ejaculates with >60% malformed morphologically live sperm [62]. Several laboratories have reported varying numbers for the average proportion of normal sperm in cats. The most frequent defects seen are malformed heads and midpiece and tightly wound tails, which show improper sperm production inside the testis [54]. The high prevalence of teratozoospermia (the presence of >60% anatomically malformed cells in the ejaculate) in Feline species has been attributed to reduced germ cell death [16].

A study by Axnér and Linde Forsberg [62] reported that male research colony cats with less than 40% morphologically normal sperm had poorer zona pellucida penetration rates than cats with more than 60% morphologically normal sperm. Due to the diminished viability of thawed sperm and the failure of hormonal ovulation induction in females, AI is difficult to achieve in Felines [63]. Pedigree cats tend to be more inbred than domestic cats, or they may be more selected for parameters other than fertility, which could have an impact on sperm morphology. In comparison to home cats, pedigree cats exhibited a lower percentage of normal sperm [62].

3.5 Sperm mitochondrial DNA integrity

Assessing mitochondrial function can predict the quality of sperm motility [64]. Mitochondria are a key source of energy for sperm movement. Often, sperm motility issues are related to defects in mitochondrial membrane integrity [50]. Furthermore, polymorphic mutations in genes encoding oxidative phosphorylation complexes and transfer ribonucleic acid of mitochondrial deoxyribonucleic acid (DNA) have been linked to reduced sperm motility [65]. A chromosome’s specific DNA sequence can be found and located using the laboratory technique known as fluorescence in situ hybridization [66]. This method involves attaching the semen’s entire set of chromosomes to a glass slide, followed by exposure to a “probe” a tiny piece of purified DNA dye-tagged with a fluorescent tag [67]. The fluorescently labeled probe locates its matching sequence within the collection of chromosomes and binds to it [66]. The chromosome and sub-chromosomal sites where the fluorescent probe bound can be observed with the aid of a specialized microscope [68].

4. Factors affecting cryopreservation of cat semen

4.1 Cold shock susceptibility

The plasma membrane’s composition affects sperm’s sensitivity to cold shock, and it varies not only between species but also between and within individuals [69]. To the authors’ knowledge, it is unknown what the Feline sperm membranes are made of. Although there was obvious acrosomal damage in one study, cooling rates of 14 and 4°C/min to 0 or 5°C did not reduce sperm motility. Nevertheless, the study only included a small number of cats in each group [70]. As demonstrated in cats where a slow cooling rate (0.5°C/min) resulted in less acrosomal damage than rapid or ultra-rapid cooling, the use of proper slow cooling procedures may be the most effective technique for minimizing sperm cold shock [71]. Sperm motility was shown to be somewhat resilient, but acrosome integrity was highly susceptible to cold shock since massive acrosomal membrane degradation did not occur along with a drop in sperm motility [72].

The ability of epididymal sperm to tolerate cold shock in comparison to ejaculated sperm was observed in a wide range of species [73, 74]. Previously, it was discovered that the same cat’s epididymal and ejaculated sperm had different morphologies. This difference might have been brought about by contact with seminal plasma [75]. Although both forms of cat sperm can be properly cooled and cryopreserved while retaining fertilizing ability, it has not been properly examined whether ejaculated and epididymal cat sperm differ in their susceptibility to cold shock [76, 77].

Due to the protective effect of egg yolk against cold shock provided by low-density lipoproteins, egg yolk is frequently used in most semen extenders. The unknown exact mechanism determines how the lipoproteins provide this protection, but it happens at the membranes outside the surface [78]. According to Glover and Watson [79], when repeated rapid cooling to 0°C of ejaculated cat semen was applied, egg yolk greatly reduced the severity of the reduction in sperm motility. Egg yolk also provided a significant level of protection against cold shock. However, a study by Glover and Watson [79] revealed that egg yolk did not improve cat sperm’s survival at 5°C. In a study by Pukazhenthi et al. [71], sperm motility was not maintained for extended periods in the presence of egg yolk for both cooled and controlled sperm, however, the impact of egg yolk on sperm membrane and acrosomal integrity was not mentioned. Moreover, Harris et al. [80] were able to show that an egg yolk semen extender improved the viability and motility of sperm when they were kept in cold storage.

4.2 Osmotic stress

To determine “optimal” chilling or cryopreservation protocols, researchers have looked at how sensitive cat sperm are to changes in osmolality and different cooling rates in terms of sperm motility loss or cell membrane damage [81]. Cat semen’s osmolality ranges from 290 to 320 mOsm/kg, and isotonic diluents are the right alternative for dilution. Moreover, the osmolality of most semen extenders is around 300 mOsm, but 800–900 mOsm is reached when cryoprotectants are added [82]. It was demonstrated that shrinkage brought on by hypertonic solutions is less harmful than swelling and membrane breakage brought on by hypotonic solutions in cat sperm [83]. However, the breakdown of the membrane and loss of sperm motility are both minimized when cryoprotectants are removed in stages using an isotonic solution because sperm motility is more susceptible to changes in osmolality than membrane integrity [83].

Although sperm from cat epididymides has been studied in numerous studies, it is still unknown what the oxidative status of sperm from the epididymis cauda looks like [84]. The effectiveness of cooling techniques, such as those for semen cryopreservation, is limited by this lack of understanding. However, it was understood that changes in the oxidative status result from the sperm’s susceptibility to reactive oxygen species (ROS) attacks [85]. This happens when the sperm cell’s cytoplasm is decreased and has fewer antioxidants, and its plasma membrane is abundant in polyunsaturated fatty acids, which ROS can more readily oxidize [86]. This oxidative damage may affect DNA integrity, lipid and protein activities, and sperm motility, and possibly lead to cellular death [87]. Understanding how the oxidative condition of the epididymal environment changes throughout numerous cooling periods may therefore enable the production of particular extenders for Feline epididymal sperm, such as the usage of antioxidants [85, 88]. When in equilibrium with these chemicals, the antioxidants counteract the negative effects of ROS [85].

4.3 Cooling rate

Mammalian semen is typically preserved using a process called cryopreservation which involves gradually cooling the sample to −196°C. According to Pukazhenthi et al. [71], slow cooling (0.5°C/min) was preferable to rapid cooling (14°C/min) for maintaining the morphological integrity of cat sperm. Before immersion in LN₂, a suitable freezing rate (+5°C to −80°C at −10°C/min) was used [89]. However, it was recently shown that a slow freezing rate of 3.85°C/min from +5°C to −40°C before immersion in LN₂ produced better sperm motility than that faster freezing rates [90].

A study by Angrimani et al. [85] showed that cooling semen to 20 and 10°C causes a decrease in sperm motility, progressive motility, and velocity traits. Feline sperm is very sensitive to low temperatures. Cooling rates of 14 and 4°C/min to 0 or 5°C did not cause a decrease in sperm motility while there was conspicuous acrosomal damage; however, the number of cats in each group was not large. To enable cells to osmotically adapt to cryoprotectants, sperm is cooled during freezing. Therefore, these results indicate that the best freezing or cooling rate for Feline sperm is yet unknown.

4.4 Addition of cryoprotectants

Ingredients added in the cryoprotectants protect sperm during cryopreservation and thawing, but may also have a toxic effect. Glycerol is frequently used as a cryoprotectant during the semen cryopreservation process. It has also, however, a negative effect on sperm because of its own toxicity and high osmolality. Equex STM paste, a detergent that improves the protective effect of egg yolk is often included in extenders for cryopreservation of dog semen and was used for cat sperm [6, 91]. Although Equex STM paste protects the acrosomes of epididymal cat sperm during freezing–thawing, it had a negative effect during post-thaw incubation [91]. Harris et al. [77] showed that only the sperm viability of ejaculated semen varied between extenders, whereas the sperm motility and viability of epididymal semen were better preserved in the presence of egg yolk than in an electrolyte-free solution.

A study by Hermansson et al. [78] showed that the temperature intervals do not appear to make Feline sperm sensitive to cold shock, most frequently used for chilling before cryopreservation or liquid sperm preservation. It is also possible to conclude that the 20% egg yolk added to the preservation extender is good for maintaining sperm motility and acrosome integrity but poor for maintaining plasma membrane integrity. Egg yolk had the same impact on both ejaculated and epididymal semen because there was no evidence of a relationship between sperm type and extender. The sperm damage seen after cat semen was cryopreserved was caused by sperm freezing and thawing protocols rather than the cooling procedure [90, 91, 92]. Therefore, rather than focusing on the chilling method, efforts should be made to develop semen extenders that protect the sperm during freezing and thawing and that prolong longevity to improve the outcomes of cat semen preservation.

5. Cryopreservation methods in wild and domestic cats

There are three methods to cryopreserve cat sperm samples (i.e., utilizing dry ice, LN₂ vapor, or a programmed biological freezer). The dry ice technique was traditionally used and normally applied for cryopreserving cat semen pellets. The frozen pellets were further cooled down and are eventually stored in LN₂ storage [93, 94]. In addition to pellet-freezing, dry ice is used to initially freeze samples loaded in 0.25 mL straws by placing the straws on dry ice for 10 min, the frozen semen straws are then stored in LN₂ [92]. In more recent studies, cryopreservation of cat semen has been conducted by placing the semen in LN₂ vapor (Table 3) which is more economical than using a programmable freezer and more convenient than using dry ice [93]. With this method, sperm-loaded vessels are placed horizontally or vertically over the surface of LN₂ for a period of time to allow the sample temperature to decrease from 4 to 5°C to approximately −40°C [90], −100°C [94], −110°C [78], −120°C [7] or − 130°C [95] before the samples are plunged in the LN₂.

Freezing package	Distance above LN2 level (cm)	Time (min)
1.5 mL cryotube	unspecified	12
0.5 mL straw	unspecified (−130°C)	15
0.25 mL straw	unspecified	15
0.25 mL straw	unspecified (−110°C)	7
0.25 mL straw	unspecified (−120°C)	10
0.25 mL straw	2	2
0.25 mL straw	4	10
0.25 mL straw	4	20
0.25 mL straw	6	15
0.25 mL straw	6	20
0.25 mL straw	7	10

Table 3.

Cryopreservation of cat sperm by using the LN₂ vapor method. Freezing vessels are placed horizontally above the LN₂ surface before being plunged into LN₂ [93].

According to previous reports, the horizontal placement is performed for samples loaded in 1.5 mL cryotubes [37] and both 0.5 mL [96] and 0.25 mL [33, 35, 97, 98] straws. The distances between the cryotubes/straws placed horizontally and the surface of the LN₂ are unchanged throughout the process [99], or 7 cm [88] above the LN₂ surface; (Table 1). With the vertical placement of samples in an LN₂ tank, the distance between the 0.25 mL straws in a canister and the LN₂ surface in a tank is altered in three steps prior to plunging the straws in LN₂ [36, 100, 101]. This technique was adapted from the method of dog semen cryopreservation, where the top of the canister was held at 7, 13, and 20 cm below the opening of the tank for 2 and 1 min, respectively [102].

Alternatively, vertical cryopreservation of cat sperm is accomplished in a specialized type of vacuum. Straws (0.25 mL) in a pre-cooled (5°C) stainless steel cylinder (14 × 4 cm) are placed in the Dewar flask (25 cm in diameter and 47 cm in height) at specific distances above the surface of LN₂ to achieve the desired cooling rates such as 3 cm above LN₂ to achieve 3.85°C/min freezing rate with the cylinder ultimately being immersed in LN₂ [90]. Using this cryopreservation technique, a slow cooling rate (3.85°C/min) resulted in greater sperm motility and normal acrosomes after thawing of cat semen as compared with the more rapid cooling rates (9, 22.8, 36, and 43°C/min, 78). In addition, multi-step LN₂ vapor cryopreservation was also conducted in the study by Vick et al. [12], by placing 0.25 mL straws above the surface of the LN₂at 7 cm for 1 min and 3.5 cm for 1 min prior to plunging the straws into LN₂. With this study, however, there was no indication when the methodologies used were reported as to whether the straws were positioned horizontally or vertically. Production of cryopreserved cat semen by using a programmable freezing system has also been reported [77, 103, 104]. On the basis of these reports, specimens submitted for cryopreservation were epididymal and ejaculated semen. The cooling rate was relatively slow during the initial stage of cryopreservation (i.e., −1°C/min from 4 to −1°C or − 25°C), to minimize ice crystal formation in the cells, and thereafter was rapidly accelerated (i.e., −30°C/min or greater) until the final temperature of −100 [103] or − 196°C [77, 104] was reached. The cryopreserved samples were subsequently transferred to an LN₂ container.

6. Thawing temperatures

The temperatures which are used for thawing play a vital r0le in post-thaw sperm survivability. According to a study by Chatdarong et al. [105], the semen of cats after thawing at 37°C for 15 sec or at 70°C for 6 sec had no effect on sperm quality. Moreover, it was recommended to reduce the toxic effect of some ingredients in the semen extender during post-thaw incubation. In contrast, Rijsselaere et al. [106] observed that rapid thawing at 60°C for 5 sec resulted in fewer sperm that displayed acrosome reaction and fewer deformed sperm heads than slow thawing at 37°C for 1 minute. Since Chatdarong et al. [105] used ejaculated semen instead of epididymal semen, different freezing procedures were used, and although both studies compared a fast thawing rate to a slower one, different temperatures and times were utilized, which may be the reason for the discrepancy.

7. Conclusion

It is crucial to develop cryopreservation techniques for domestic cat semen in order to preserve it as well as to use the current information/data from these studies to create and improve protocols for the preservation of wild Felid semen. Each cryopreservation procedure needs to be carefully examined/assessed in order to provide frozen–thawed samples of promising quality. The current contribution compiles earlier works on cat semen cryopreservation and outlines the cryopreservation procedures in a step-by-step fashion, starting with the semen source and ending with post-thaw dilution. Semen can be collected from cats by using an artificial vagina and electro-ejaculation or collected from the epididymides by using manual compression, flushing, cutting/mincing/slicing, and puncturing.

The global decline in the number of domestic and wild cats necessitates using conservation measures and establishing germplasm banks [18]. Furthermore, the establishment of germplasm banks aims to prevent the negative effects of diminished genetic diversity on threatened populations by preserving biological samples from various species [63]. Moreover, the establishment of germplasm banks would be aided by the creation of efficient semen cryopreservation protocols. The pregnancy rates after insemination with frozen–thawed sperm have been widely variable, ranging from about 10–50% compared unfavorably to 70–80% of fresh semen. This shows that the cryopreservation process invariably causes cryoinjuries and negatively affects sperm viability and fertilizing ability [106].

Acknowledgments

The Agricultural Research Council, Animal Production (Germplasm Conservation and Reproductive Biotechnologies) colleagues are acknowledged for their support.

Conflict of interest

The authors declare no conflict of interest.