PRESERVATION OF DOG SEMEN

PRESERVATION OF DOG SEMEN

Preserving and storing dog semen is a valuable practice in canine reproduction, offering numerous advantages for breeders and dog enthusiasts. This article explores the various methods and considerations involved in the preservation of dog semen, emphasizing the significance of reproductive technologies in maintaining genetic diversity and achieving breeding goals.

importance of dogs in our lives

The first recorded and successful artificial insemination (AI) was performed in the dog in 1785 by Spallanzani, the first successful use of frozen canine semen was reported by Seager in 1969. However, only in the last two decades AI in the dog became a rather widespread and generally accepted breeding technique. It may be applied for medical reasons, to prevent infections, to overcome travel and quarantine restrictions and to avoid long, expensive and stressful travels with the animals. In conjunction with cryopreservation AI is especially indicated to build up stocks from excellent studs and for conservation of semen from rare breeds. Only semen meeting the quality criteria outlined in table 1 should be used for conservation. The same quality criteria apply when studs are examined for breeding soundness.

Dogs have been an integral part of our society since time immemorial. The history of the relationship  between human and dog started from hunting wild animals for food and went through herding to being a companion, a guide for blind people, a part of police squad, and many more. Globally, there are different sorts of dog breeds serving different purposes. However, in the past few decades, the need for dogs as a companion animal has increased several folds and the emerging societal changes for the desire of exotic breeds have led to the transportation of dogs over a long distance which has popped up into a cumbersome and costly task. Thereby experiments of cross breeding and artificial insemination are taking place for an awfully long time in dogs and effecting tremendous progress in the field of canine reproduction. However, in canine reproduction particularly, artificial insemination has opened up a new world of breeding possibilities and allowed the breeder to impregnate the medically disabled bitch with the procured desirable semen over the globe. This modern reproductive technology also caused a reduction in sexually transmitted diseases and the stress level of the animals attributed to transportation for mating. Meanwhile, crossbreeding has heralded development of new breeds by exploiting the germplasm of selective breeds to the fullest and at the same time some other native breeds face extinction due to negligence which urge into a huge demand to preserve the germplasm before they vanish from the face of the earth. On the other hand, till date most of the crossbreeding is limited to natural mating due to lack of scientific knowledge of artificial insemination among the dog owners and availability of fresh and chilled semen. The fresh and the chilled semen can be stored for a limited period of days. Considering all the above factors, cryopreservation of dog semen could be an easy, cost-effective way that may allow worldwide access and availability of the frozen semen from sires for a longer period even after they decease (Pezo et al., 2017).

MYSTERY AND IMPLEMENTATION OF CRYOPRESERVATION

Cryopreservation is the third-generation technique which facilitates preservation of good quality semen for a longer period, and ensures easy and cost-effective transport of desired semen over long distances and preservation of good quality germplasm of a stud for years together (Kutzler et al., 2005). Briefly, it is a process of freezing semen at -196 ºC in liquid nitrogen and can be used later, whenever required. However, in this technique, there are some specific requirements that must be fulfilled for a successful cryopreservation, as the purpose is not merely storing the semen sample but also maintaining the fertilizing capacity of the sperm and utilizing it later for successful fertilization.

Prior to cryopreservation, the semen ejaculate must be analysed for certain characteristics to assess whether it is fit for use or not. This includes routine semen analysis, viz. macroscopic and microscopic tests like volume, colour, consistency, initial motility, mass motility, pH and concentration. Apart from these, there are a wide variety of other advanced evaluation methods available to assess the semen quality more precisely like Hypo-Osmotic swelling test (HOST), Zona Pellucida binding Assay (ZBA), fluorescent probes and flow cytometry and computer assisted sperm analysis (CASA). After the semen is declared fit for further processing, it is extended with the dilutor. Composition of a dilutor/extender needs to be calibrated precisely. Even a small change in the constituents can gravely affect the whole process. Basically, a dilutor is composed of water as a solvent, buffer to regulate pH, sugar as an energy source, an organic material like egg yolk or milk to prevent cold shock, a cryopreservative like glycerol or DMSO, an antibiotic to control microbial growth and additives to improve fertility. Dog semen is composed of three portions. The first portion mainly comprises of prostate secretion with an admixture of epithelial cells, urine, bacteria, and sperm cells; the second portion varies from 0.5 to 2 mL and is sperm-rich and the last portion comprises mainly of prostatic gland secretion with a volume variable up to 30 ml (Kutzler et al., 2005). Seminal plasma is considered detrimental to survivability of sperm during cryopreservation. Hence, seminal plasma must be removed by centrifugation and discarding the supernatant. The sediment consisting of sperm cells is utilized for further processing that includes extension, packaging, equilibration at 4 °С for 4 hours, freezing and storing.

“Timing is everything……………” WHEN TO DO ARTIFICIAL INSEMINATION?

Bitches are mono-estrous animals, i.e., they come in heat once or twice a year. Due to this peculiarity, the correct timing of insemination is particularly important. The bitch usually presents a relatively long follicular phase and there is considerable variability in the onset of estrus behaviour and acceptance of the male, making it difficult to determine occurrence of the LH surge and onset of ovulation. However, there are several tests available to determine appropriate timing of insemination like vaginal cytology, progesterone estimation, vaginal endoscopy and ultrasonography (Wright, 1990). Generally, ovulation occurs 48 hours after preovulatory LH peak. Therefore, careful planning and synchronizing of both the time of mating and ovulation can be a key step in successful canine artificial insemination. Under such circumstances, frozen/thawed semen is found to be more advantageous compared to fresh and chilled semen. Basically, insemination with fresh / chilled semen needs to be performed on the day of ovulation, and a second insemination 2 days later; whereas insemination with frozen/thawed semen is performed 2 days after ovulation, and the second insemination 48 h later, considering the time required for premature oocytes to mature in the oviducts (Linde-Forsberg, 1995).

Semen preservation and dose for insemination

Except for transfer of native semen only the sperm rich fraction should be used for semen preservation.

Native Semen

Native semen may be transferred due to medical or behavioural reasons without any further handling. The ejaculate may be split, however, transfer should be performed as soon as possible after semen collection and evaluation, although fertilization rates of 70% have been observed with unextended canine semen after two days of storage at 4°C (Tsutsui, 2002).

Table 1. quality criteria for dog semen (Hoffmann, 2003)

forward motility	> 75 %
pathomorphology	< 20 %
pH-value	6.2-7.2
total number of spermatozoa	> 0.3-1.0 x 106 (1)
volume	0.5-2.0 (2)

1. depending on breed; e.g., Dachshund >0.3; Dobermann >1.0
   2. Sperm rich fraction

Chilled Semen

The use of chilled semen requests the availability of the donor on a short call and semen transport within a short term. A number of semen extenders has been described; generally accepted is a tris-buffered solution containing 20% egg yolk, 12.5% fructose and an admixture of an antibiotic. The cooling down to about 5°C is over a period of 2 hours. Depending on the concentration of spermatozoa, the ratio (volume) of ejaculate to extender may vary between 1:3 to 1:5. Mild centrifugation might become necessary when a selective collection of the sperm rich fraction was not possible. The total dose is stored in an appropriate sealed vial. The whole sperm rich fraction may be extended and used as one dose. If the ejaculate has to be split, one dose should at least contain 100-200×10⁶ spermatozoa (Linde-Forsberg, 1991). The fertilization capacity of chilled semen decreases progressively, and it may be difficult to estimate the time after which the use of a certain semen portion may become unpromising, as after 5 days of storage motility may be still high, when fertilization capacity has considerably decreased (Tsutsui et al., 2002).

Frozen Semen

Whereas pregnancies may be obtained with fresh semen of inferior quality, albeit with smaller litter sizes, insemination of frozen semen of poor quality is unpromising. Thus, raw material of adequate quality and appropriate preservation procedures are indispensable for acceptable results. There are various protocols for cryopreservation of canine semen. Basically the same extender as used for the preparation of chilled semen may be used. The concentration of the cryoprotectant glycerin is at 6%. In our clinic semen meeting the criteria given above is diluted and packed in 0.5 ml straws. Following cooling to 5°C over 2 hours they are left for 10 min at-140°C (vapor of liquid nitrogen). Final storage is at-196°C. It is an absolute requirement to test the quality of each individual batch by thawing (37°C; 20 seconds) and reexamining a frozen aliquot, as the suitability for freezing may vary considerably between different ejaculates of an individual stud. Loss in forward motility should not exceed 20%, pathomorphology may be increased up to 30% (Hoffmann 2003). For insemination, a number of straws corresponding to approximately 1.5×10⁸ progressively motile spermatozoa is thawed and pooled.

Insemination

Insemination regime

As with natural mating, precise determination of the optimal time of insemination is less critical using native or chilled semen compared to cryopreserved semen due to the longevity of fresh canine spermatozoa in the female genital tract which is between 5-7 days, yielding a broad biological leeway for pregnancy even under suboptimal insemination management (Jeffcoate, Lindsay, 1989). Thus, using native or freshly provided chilled semen, a single insemination around the time of ovulation may be sufficient, although significantly higher pregnancy rates have been found with two inseminations (Linde-Forsberg et al., 1993). In contrast, the limited life-span of frozen-thawed spermatozoa in the female genital tract, which is estimated to be shorter than 12-24 hours (Concannon et al., 1989), requires a precise definition of the time of insemination in the individual bitch, which is about 2-5 days after ovulation due to a comparatively long postovulatory process of oocyte maturation. Repeating the AI after 24-48 hours may result in significantly higher pregnancy rates and litter sizes (Tsumagari et al., 2003) probably due to the occurrence of an extended ovulation and maturation period. Indications for the appropriate time of insemination may be obtained by observation of behavioural changes, vaginal cytology, vaginoscopy and determination of the electrical impedance. Repetitive examinations are necessary and the variability inherent to these approaches and the underlying parameters is high. It is therefore recommended to directly determine the underlying hormonal changes by applying a reliable quantitative assay for progesterone. Progesterone levels closely reflect the pre and postovulatory luteinization of granulosa cells with about 1-2 ng/ml during the LH-peak and about 5 ng/ml at ovulation. Termination of oocytes maturation is more difficult to assess due to the variability in the increase of progesterone levels with formation of the corpora lutea. However, progesterone levels between 10-12 and even up to 20 ng/ml are generally considered as optimal for insemination with cryopreserved semen (Linde-Forsberg et al., 1989).

Technique of semen transfer

Due to the anatomical structure of the vagina intrauterine insemination in the dog is more difficult than in other species and requires quite some experience. This is largely due to a dorsal median fold of tissue that extends caudally from the vaginal portion of the cervix. At vaginoscopy the caudal portion of the fold and the constrictions of the lateral and ventral vaginal walls give a distinct appearance, referred to as pseudocervix. The true vaginal portion of the cervix is cranial to the pseudocervix. This makes intrauterine cannulation difficult, also because the cervical canal is nearly perpendicular to the longitudinal axis of the vagina and the uterine body.

Currently four methods are used for semen transfer in dogs: deep intravaginal insemination, transcervical insemination using an rigid catheter (Norwegian Catheter) with transabdominal fixation of the cervix, transcervical endoscope-aided insemination and intrauterine insemination using a laparoscopic approach. Freshly ejaculated spermatozoa and spermatozoa from chilled semen may readily penetrate the cervix and reach the site of fertilization in the oviduct. Hence deep intravaginal insemination with this type of semen is common practice. As a substitute of the vaginal plug by the erected bulbus glandis it has been generally recommended to elevate the rear end of the bitch at a 45° to 60° angle for 5-20 minutes after AI. However, pregnancy rates and litter size was not different comparing elevation times of 1 and 10 min (Pinto et al., 1998). In respect to cryopreserved semen it was widely accepted that semen deposition must be intrauterine for optimal results. However, data from literature are contradictory as in some studies no significant difference was found between precervical and transcervical insemination using frozen thawed semen (Rota et al., 1999). Yet up to now the statistical basis is very weak not allowing any definite conclusions (see below). Hence we still recommend intrauterine semen deposition using the transcervical approach since the laparoscopic approach is not accepted by many pet owners, which consider this method as unethical and too risky for the bitch. Yet it must be anticipated that the transcervical approach may be difficult in maiden dogs and in those cases with a high discharge of cervical mucus, blinding the optical system. In these cases the semen should be placed as close as possible to the orificium externum of the cervix.

HOW TO DO ARTIFICIAL INSEMINATION?

Packaged straws or pellets are thawed prior to artificial insemination at 70 ºC for 8 seconds or 37 ºC for 30 seconds. Insemination can be done either deep vaginal or intra uterine. For deep vaginal insemination, a simple plastic catheter with disposable syringe can be utilized or commercial catheters are also available. On the other hand, intrauterine insemination can be done surgically or non-surgically. Non-surgical method is like the deep vaginal method involving use of an outer plastic catheter and an inner metal catheter as a guide or alternatively, an endoscope-assisted vaginoscopic method can also be employed using a cysto-uretheroscope. Surgical method of intrauterine insemination is considered unethical and hence not used for the purpose. It involves laparoscopy and laparotomy after induction of anaesthesia. Intrauterine insemination is more successful compared to deep vaginal insemination with frozen semen.

Pregnancy rates and litter sizes

Success of AI in dogs depends on various parameters such as type and quality of semen used, sperm dosage, the method and site of semen deposition, insemination regime and breed.

Provided semen of adequate quality is used, success of AI using native or chilled semen is equal to that after natural mating (Pinto et al., 1999).

Several studies are available reporting pregnancy rates and litter sizes after AI with frozen-thawed semen in bitches using intravaginal and/or intrauterine insemination. However, results vary substantially obviously due to the high variability of the conditions for insemination. However, in a relatively large comprehensive study using semen of defined quality, pregnancy rates/liter size after a single insemination per cycle were 84.3%/5.0 ± 3.0 puppies after intrauterine semen deposition, but only 34.8% / 2.5 ± 1.3 puppies after intravaginal insemination. These data show that–provided a good method of cryopreservation and intrauterine semen deposition are applied–results with AI using frozen-thawed semen may be comparable to those obtained with natural mating. In those studies yielding no difference between intravaginal vs. intrauterine insemination, the benefit of intrauterine insemination may be overridden by higher doses of spermatozoa or by higher numbers of AI’s per cycle. Accordingly, in the above mentioned study whelping rate/litter size after intravaginal deposition of frozen-thawed semen increased to 63.9%/5.1 ± 3.1 when three inseminations per cycle were applied (Linde-Forsberg et al., 1999).

Importance of Semen Preservation:

Genetic Diversity:

• Conserving Breeds: Semen preservation enables the conservation of rare and valuable dog breeds.
• Avoiding Genetic Drift: Preserving semen allows breeders to maintain genetic diversity and prevent the narrowing of gene pools.

Global Reproductive Programs:

• International Breeding: Semen preservation facilitates international breeding programs by overcoming geographical barriers.
• Importance for Rare Breeds: For rare or endangered breeds, semen preservation becomes essential for global conservation efforts.

Methods of Semen Preservation:

Refrigeration (Short-Term Storage):

• Temperature Control: Semen is stored at refrigeration temperatures (2-8°C).
• Duration: Typically viable for a few days.
• Advantages: Suitable for short-term transportation and insemination.

Cryopreservation (Long-Term Storage):

• Freezing Semen: Semen is frozen using cryoprotectants and liquid nitrogen.
• Duration: Can be stored for extended periods (years).
• Advantages: Allows long-term storage, making it ideal for future breeding seasons or shipping internationally.

Semen Extenders:

• Dilution for Preservation: Extenders are added to semen for dilution, aiding in both refrigeration and freezing processes.
• Protective Agents: Extenders contain substances to protect sperm during storage.

Factors Affecting Semen Quality:

Semen Collection Techniques:

• Artificial Insemination (AI): Proper collection techniques for AI ensure the collection of high-quality semen.
• Stress Reduction: Minimizing stress during collection positively influences semen quality.

2. Semen Processing:

• Centrifugation: Techniques such as centrifugation separate seminal plasma, enhancing sperm concentration.
• Removal of Debris: Removing debris and non-sperm components improves the quality of preserved semen.

Semen Evaluation:

• Quality Assessment: Regular evaluation of sperm motility, concentration, and morphology ensures the quality of preserved semen.
• Viability Testing: Assessing sperm viability is crucial for successful insemination.

Practical Considerations:

Health Screening:

• Disease-Free Semen: Ensuring that the donor dog is free from sexually transmitted diseases.
• Genetic Testing: Screening for genetic disorders contributes to the overall health of the breeding program.

Proper Documentation:

• Accurate Records: Maintaining detailed records of each semen sample, including the donor’s information, date of collection, and processing details.
• Traceability: Documentation aids in traceability and pedigree management.

Legal and Ethical Considerations:

• Ownership Rights: Clarifying ownership rights and responsibilities regarding stored semen.
• Ethical Practices: Adhering to ethical standards in semen preservation and storage.

Conclusion:

Semen preservation is a crucial aspect of modern canine breeding, offering breeders unprecedented opportunities to conserve genetic diversity, overcome geographical constraints, and contribute to the welfare of rare and endangered breeds. As technological advancements continue to refine preservation methods, the responsible use of semen storage aligns with the broader goals of ensuring the health, diversity, and sustainability of canine populations worldwide. Whether for breeding purposes or conservation efforts, the preservation of dog semen stands as a testament to the evolving landscape of reproductive technologies in the realm of canine reproduction.Success with artificial insemination of frozen dog semen has been limited and the sublimed reasons are associated with ordered logical studies of cryopreservation and identification of the exact time of fertilization in dog.  Therefore, this article is to highlight the neglected areas of cryopreservation in dog and creating awareness among dog breeders and researchers for adapting new and advanced technologies for better and safe utilization of the limited gene pool to preserve the varied breeds, and to reciprocate all the love and loyalty to our little friends.

 Compiled  & Shared by- This paper is a compilation of groupwork provided by the

Team, LITD (Livestock Institute of Training & Development)

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ARTIFICIAL INSEMINATION IN DOG:PRACTICES & REGULATION FOR IMPORT OF CANINE SEMEN IN INDIA