EFFECT OF DILUENT, STORAGE TIME AND TEMPERATURE ON EQUINE EPIDIDYMAL SPERM

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

Faculty of Veterinary Medicine

Anna Ellung Lehmann

EFFECT OF DILUENT, STORAGE TIME AND TEMPERATURE ON EQUINE EPIDIDYMAL SPERM

QUALITY PARAMETERS

SPERMOS SKIEDIKLIO, LAIKYMO TRUKMĖS IR TEMPERATŪROS ĮTAKA ŽIRGŲ ANTSĖKLIDŽIO

SPERMATOZOIDŲ KOKYBĖS RODIKLIAMS

MASTER THESIS

of Integrated Studies of Veterinary Medicine

Supervisor: Dr.

Neringa Sutkevičienė

KAUNAS 2019

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THE WORK WAS DONE IN THE DEPARTMENT OF LARGE ANIMAL CLINC CONFIRMATION OF THE INDEPENDENCE OF DONE WORK

I confirm that the presented Master Thesis “Effect of diluent, storage time and temperature on equine epididymal sperm quality parameters”

1. has been done by me;

2. has not been used in any other Lithuanian or foreign university;

3. I have not used any other sources not indicated in the work and I present the complete list of the used literature.

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TABLE OF CONTENTS SANTRAUKA ... 5

SUMMARY ... 6

INTRODUCTION ... 7

1. LITERATURE REVIEW ... 8

1.1 Equine male reproductive organs ... 8

1.2 The Epididymis ... 9

1.2.1 Anatomy of epididymis ... 9

1.2.2 Physiology of epididymis ... 9

1.3 THE MORPHOLOGY OF STALLION SPERM ... 9

1.3.1 The sperm head ... 9

1.3.2 The sperm tail ... 10

1.4 EQUINE SPERMATOGENESIS ... 10

1.5 Fertility of fresh and frozen epididymal sperm ... 11

1.6 Extraction/harvesting of epididymal sperm ... 11

1.7 Andrological Examination ... 13

1.7.1 Morphological Examination of external Genitalia ... 13

1.7.2 Macroscopic semen evaluation ... 14

1.7.3 Microscopic semen evaluation ... 14

1.7.3.1 Subjective motility determination ... 14

1.7.3.2 Computer-assisted sperm analysis ... 14

1.7.3.3 Concentration and total sperm count ... 15

1.7.3.4 Morphology ... 15

1.7.3.5 Viability ... 16

1.7.3.6 Foreign Cell Count ... 16

1.7.3.7 Agglutination ... 17

1.8 Dilution and conservation of spermatozoa ... 17

1.8.1 Preparation of equine spermatozoa with fresh semen diluent ... 17

1.8.2 Centrifugation ... 18

1.8.3 Cooling of fresh stallion semen ... 19

1.8.4 Cryopreservation of stallion semen ... 19

2. RESEARCH METHODS AND MATERIALS ... 20

2.1 Motility assessment ... 23

2.2 Viability assessment ... 23

2.3 PH evaluation ... 24

2.4 Sperm concentration ... 25

2.5 Morphological evaluation ... 25

2.6 Statistical analysis ... 26

3. RESEARCH RESULTS ... 27

3.1 Descriptive statistics ... 27

3.1.1 Motility in two different diluents ... 27

3.1.2 Viability and pH in two different diluents ... 27

3.1.3 Concentration in 2 different diluents ... 28

3.1.4 Motility at two different temperatures ... 28

3.1.5 Viability and pH at two different temperatures ... 29

3.2 Sperm quality values at 24, 48 and 72 hours ... 30

3.2.1

Subjective motility in different diluents ... 30

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3.2.2 SCA motility in different diluents ... 31

3.2.3 Progressive motility ... 32

3.2.4 Viability ... 33

3.2.5 pH values ... 34

3.2.6 pH of 2 diluents, at 4 and 20°C on 3 days ... 35

3.3 Morphology of Epididymis ... 36

4. DISCUSSION OF RESULTS ... 40

CONCLUSIONS ... 41

RECOMMENDATION ... 42

ACKNOWLEDGEMENT ... 43

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SPERMOS SKIEDIKLIO, LAIKYMO TRUKMĖS IR TEMPERATŪROS ĮTAKA ŽIRGŲ ANTSĖKLIDŽIO SPERMATOZOIDŲ KOKYBĖS RADIKLIAMS

Anna Ellung Lehmann

Magistro baigiamasis darbas

SANTRAUKA

Spermos paėmimas iš antsėklidžio gali būti paskutinė galimybė išsaugoti eržilo genetinius duomenis po neplanuotos jo kastracijos arba mirties.

Šio tyrimo tikslas buvo ištirti spermos skiediklio, laikymo trukmės ir temperatūros įtaką žirgų antsėklidžio spermos kokybei. Sperma buvo tiriama Lietuvos sveikatos mokslo universiteto (LSMU) Stambiųjų gyvūnų klinikos Gyvūnų reprodukcijos laboratorijoje. Antsėklidžio sperma buvo surenkama iš Stambiųjų gyvūnų klinikoje (LSMU) kastruotų eržilų.

Iš viso, nuo 2018 metų gruodžio mėn. iki 2019 metų gegužės mėn., buvo surinkta 8 kastruojamų žirgų, kurių amžius nuo 3 iki 10 metų, antsėklidžio sperma. Equiplus ir Ringer B Braun tirpalai buvo naudojami kaip antsėklidžio spermos skiedikliai. Mėginių laikymo temperatūra buvo 4 °C ir 20 °C, spermos kokybės parametrų analizė buvo atlikta iškart po pristatymo į laboratoriją ir taip pat po 24, 48 ir 72 valandų. Progresyvus ir bendras spermatozoidų judrumas buvo matuojamas subjektyviai ir naudojant SCA. Mėginiuose, skiestuose Equiplus skiedikliu ir laikomuose 4 °C temperatūroje, nustatytas didžiausias spermatozoidų judrumas tiriant visais trimis nustatymo būdais (23,3±4,6 proc. subjektyviai nustatyto, 64,5±5,4 proc. SCA ir 16,65±2,8 proc.

progresyvaus judrumo). PH vertė 6,56 buvo didžiausia mėginiuose su Equiplus skiedikliu esant 4

°C. Per pirmąsias 24 valandas statistiškai reikšmingo skirtumo tarp judrumo ar gyvybingumo parametrų, skirtinguose skiedikliuose, nepastebėta (p>0,05). Didžiausias spermatozoidų gyvybingumas (80,35±3,5 proc.) po 72 valandų buvo nustatytas Equiplus skiedikliu skiestuose ir 4

°C temperatūroje laikomuose mėginiuose. Žemiausias gyvybingumas (66,8±5,4 proc.) buvo rastas mėginiuose su Ringer tirpalu po 72 valandų 20 °C temperatūroje.

Spermatozoidų galvučių, paimtų iš antsėklidžio galvutės, kūnelio ir uodegėlės, morfologiniai rodikliai statistiškai nesiskyrė (p>0,05).

Raktažodžiai: žirgai, antsėklidis, spermatozoidai, judrumas, gyvybingumas, pH, morfologija

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EFFECT OF DILUENT, STORAGE TIME AND TEMPERATURE ON EQUINE EPIDIDYMAL SPERM QUALITY PARAMETERS

Anna Ellung Lehmann

Master Thesis

SUMMARY

The retrieval of sperm from the epididymis can be the last chance to save a stallion’s genetic material after unexpected castration or even death.

The objective of this study was to analyze the effect of diluent, storage time and temperature on equine epididymal sperm quality. Sperm analysis was carried out in the Animal Reproduction Laboratory of Large Animal Clinic Department at the Lithuanian University of Health Science (LUHS). The epididymal sperm was collected from castrated stallion testes in the Large animal clinic at LUHS.

In total 8 horses between the age of 3 and 10 years from December 2018 till May 2019 were castrated and epididymal sperm was collected. Equiplus extender and Ringer B Braun solution were used as diluents for the epididymal sperm samples, storage temperatures were 4 °C and 20 °C and sperm quality parameter analysis was carried out right after arrival in the Laboratory as well as after 24, 48 and 72 hours. Total and progressive sperm motility was measured subjectively as well as with SCA. Samples of Equiplus at 4 °C showed highest motility rates with all three measuring techniques (23.3±4.6% subjective, 64.5±5.4% SCA, 16.65±2.8% progressive). The pH value of 6.56 was highest in Equiplus at 4 °C. In the first 24 hours no significant (p>0.05) difference in motility, or viability were seen. Greatest viability with 80.35±3.5% of spermatozoa after 72 hours was detected in Equiplus at 4 °C storage temperature. Most unfitting viability of 66.8±5.4% were detected in Ringer solution after 72 hours at 20 °C storage temperature.

Sperm head morphology showed no significant (p>0.05) differences between caput, corpus and cauda epididymis.

Keywords: equine, epididymis, spermatozoa, motility, viability, pH, morphology

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INTRODUCTION

The death of a valued stallion can be devastating and unexpected, as can a castration be necessary for the stallions health and wellbeing. Collection of sperm from the epididymis can be the last option to secure the stallion’s genetics. But also for the survival of endangered species collection of epididymal sperm is important. The collection of epididymal spermatozoa has been reported in multiple different species(1–4). In equine breeding conservation of epididymal sperm has become more popular (5,6). One of the reasons may be that Stallion sperm performs often poorly at cryopreservation as to why cooled storage may be preferred (7). There are many commercial extenders available for semen storage. The results of particular extenders differ widely, thus choosing a suitable extender is important for successful breeding (8). The results regarding motility of epididymal and ejaculated sperm in stallions have been found to be comparable (9). The epididymis is crucial for sperm maturation and sperm storage (10). Nevertheless sperm retrieved from epididymis is fertile and a mare was impregnated with cryopreserved sperm first reported in 1957 by Barker (11). The estimated capacity of spermatozoa stored in the epididymis corresponds to 10 ejaculates (12,13).

The objective of this study was to research and analyse the effect of diluent, storage time and temperature on equine epididymal sperm quality.

Tasks of the work:

1. To analyse and compare epididymal sperm motility in different diluents, temperatures and storage time.

2. To analyse and compare epididymal sperm viability in different diluents, temperatures and storage time.

3. To analyse and compare epididymal sperm pH in different diluents, temperatures, and storage time.

4. To analyse and compare sperm morphology collected from caput, corpus and cauda

epididymis.

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1. LITERATURE REVIEW

1.1 Equine male reproductive organs

The stallion, just as other mammals, has a pair of testes located in the inguinal region, surrounded by a scrotum. In the testes is the production of spermatozoa and the hormone testosterone. The testis is positioned in a nearly horizontal plane, has an oval shape with its sides lightly compressed. Size of the testes vary greatly with an average diameter of 50 – 80 mm and a length of 80 – 140 mm (14). The testes are divided into lobules called lobuli testis. The seminiferous tubules and the interstitial tissue are found in the parenchyma of the testes. The sertoli cells have a crucial role in germ cell differentiation and isolating them but the germ cells are the most outstanding component of the seminiferous epithelium. The blood-testis barrier isolates these germ cells from the immune system of the stallion. The testes are surrounded by the tunica albuginea, which is connected to the outer surface of the visceral tunica vaginalis. The copulation organ of the stallion is the penis, supported by the prepuce. It consists of three regions: the root, attaching to the skeletal system; the body or shaft, the largest part; and the gland penis, which is the enlarged end of the penis. The penis furthermore consists of the corpus cavernosum penis and corpus spongiosum penis, which are the functional parts of the penis, as well as the urethra, bulbospongiosus muscle and associated blood vessels and nerves. Other reproductive organs and structures are located within the abdominal cavity. The spermatic cord, ductus deferens, ampulla, vesicular gland and bulbourethral gland come in pairs but the prostate gland is single with two narrow lobes (14).

Fig. 1: Anatomy of the Stallion Testis, lateral view.

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1.2 The Epididymis

1.2.1 Anatomy of epididymis

Located in the scrotum, the epididymis is a set of two, each connected to the testes and spermatic cord. The epididymis can be macroscopically divided into head (Caput epididymidis), body (Corpus epididymidis) and tail (Cauda epididymidis). Head and body of epididymis are attached dorsolateral to the testis. The head of the epididymis is flat and placed on the cranial pole of the testis. The tail of the epididymis is large, bulbous, and placed on the caudal pole of the testis.

Within the head of epididymis, the 13 – 15 efferent ducts leading from the rete testis are fusing into a single duct, the epididymal duct. The epididymal duct is coiled, approximately 45 m long and continues throughout the epididymis into the deferent duct (14–16).

1.2.2 Physiology of epididymis

The anatomical division of epididymis into head, body and tail does not correlate with the cytologic and biochemical processes of the epididymis (16). The different cells of the epididymis influence the composition of epididymal secretion and by that the environment of the spermatozoa during maturation. Through regionally different expression profiles of the proteins the microenvironment changes in the different segments (17). During maturation spermatozoa gain the ability of motility (18), recognize the zona pellucida, bind to it and fuse with the membranes of the oocyte (19).

1.3 THE MORPHOLOGY OF STALLION SPERM

The equine spermatozoon is structurally similar to that of other mammalian spermatozoa, such as the bulls, ram, boar, dog or human. With an average length of 61-86 µm (14), the size of the stallion sperm compared to its body size is significantly smaller in contrast to some other species.

1.3.1 The sperm head

It consists of a nucleus and an overlying acrosome. The acrosome contains enzymes, of which

two main ones, are acrosin and hyaluronidase. The head can be subdivided, starting cranially, into

acrosomal region, equatorial segment, post-acrosomal region and a posterior ring. The posterior

ring connects the head to the tail of the sperm. The heads shape is described as splatulate-shaped,

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flattened in its dorsoventral side. The males’ parental genetic material consists of a haploid set of somatic chromosomes as well as a sex-determining chromosome, which are located in the sperm heads nucleus. The nucleus is isolated from the cytoplasm by two lipid bilayers. The acrosome can be further divided into a connected outer and inner acrosomal membrane. It forms the acrosomal matrix in its middle and covers two-thirds of its rostral head (14). The acrosomal matrix as well as its inner membrane contain active molecules such as protein receptors and hydrolytic enzymes. The cytoplasm of the sperm head contains cytoskeletal proteins (14,15).

1.3.2 The sperm tail

The sperm tail (flagellum) is subdivided, starting cranially, into a connecting piece, midpiece, principal piece and endpiece. The axoneme, the outer dense fibers, a fibrous sheet and a mitochondria form the sperm tails functional units. The attaching part of the flagellum to the head is mainly the capitulum, segmented columns of fibers and the proximal and distal centrioles. A plasma membrane is covering the whole sperm tail (14).

1.4 EQUINE SPERMATOGENESIS

Spermatogenesis can be divided into spermatocytogenesis, meiosis and spermiogenesis. The

production of male gametes, multiplication and differentiation of germ cells, as well as the release

of spermatozoa from the sertoli cells are completed in spermatogenesis (20). Leydig cells located in

the interstitial space of seminiferous tubules produce and release the hormone testosterone, which is

needed to stimulate the diploid spermatogonium to differentiate into two diploid primary

spermatocytes by mitosis. The primary spermatocyte can then undergo meiosis I, producing two

haploid secondary spermatocytes and migrate from the exterior base of the seminiferous tubules

towards its lumen. The secondary spermatocytes can afterwards go through meiosis II, during

which each spermatocyte will produce two haploid spermatids (18). During spermiogenesis through

the interaction of spermatids with sertoli cells the spermatids can differentiate into sperm cells. By

the time the spermatozoa are created they will have finally reached the lumen of the seminiferous

tubules and can move further into the epididymis to fully mature. Most spermatozoa first gain their

motility and fertility during their transfer through the epididymis (21).

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1.5 Fertility of fresh and frozen epididymal sperm

In 1957 the first successful artificial insemination with frozen semen in a mare was accomplished by Barker and Gandier (11). The Spermatozoa were obtained after a castration of a two-year-old stallion from the epididymis. Fertility of epididymal stallion sperm was thereby proven for the first time.

Later a study was conducted by Morris (22), which examined the in vivo fertility of epididymal sperm in the stallion. Seven different insemination techniques were used on 83 mares.

The most successful conception rate of 45% (9/20) was achieved with a concentration of 200 million fresh epididymal sperm. The freezing and thawing process of epididymal sperm caused a decrease of the conception rate by 18% (9/51). After thawing the addition of Tyrode’s albumin lactate pyruvate (TALP) increased the conception rate from 0 % to 29 % (7/24).

In a study conducted by Melo and Papa (23) pregnancy rates of 69.23% (9/13) with frozen epididymal sperm and 66.6% (12/18) with fresh epididymal sperm were achieved.

Another study investigated the different pregnancy rates between fresh ejaculated semen, fresh epididymal sperm, fresh epididymal sperm with added seminal plasma and without added seminal plasma as well as frozen ejaculated semen and frozen epididymal sperm with and without seminal plasma. The study showed the best pregnancy rates with 75% (9/12) in fresh epididymal sperm with seminal plasma, suggesting that the addition of seminal plasma acts positively on the pregnancy rates (24).

Monteiro et al. compared the fertility of ejaculated and epididymal sperm after freezing and thawing process. Semen was taken from stallions, after which they were castrated and semen was extracted from the epididymis. One epididymis was stored for 24 hours at +5 °C before the sperm was obtained. The ejaculated and epididymal sperm was frozen and thawn in the same manner. A conception rate of 61.5 % (8/13) with ejaculated sperm, 92.3 % (12/13) with immediately processed epididymal sperm and 61.5 % (8/13) with epididymal sperm which was stored for 24 hours prior to sperm collection (9).

Another study evaluated the conception rates of epididymal sperm, which was collected after storage time of 24 and 48 hours at 5 °C (25). The results showed a 53.8 % (7/13) pregnancy rate in epididymal sperm stored for 24 hours and 40 % (4/10) in epididymal sperm stored for 48 hours.

1.6 Extraction/harvesting of epididymal sperm

Often the epididymal sperm is needed as the last chance of a retrieval of the stallions’ genes

by its sperm after its death. This is possible because the epididymis has the capability to store

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sperm in itself and can be extracted from it. Either the testes, together with the epididymis and vas deferens, are harvested prior to euthanasia or right after the stallions’ death. The castration can be performed as a routine castration would be done, merely it should be taken into consideration to leave as much of the vas deferens as possible attached to the removed testis and epididymis. The vas deferens should be ligated near the cut to prevent leakage of sperm during shipment and handling. After the removal of the testes from the stallion they should be washed with 37°C sterile lactated Ringer’s solution. For transportation the tissues are placed in a sterile plastic bag and vacuum sealed, 5 ml sterile lactated Ringer’s solution may be added to ensure no drying out of the tissues. A second bag may be added for an extra layer of protection. A shipping container suitable for fresh cooled equine semen will keep the testes at an adequate temperature during transportation.

When adding frozen coolant material, it should be separated from the tissues by a towel or packing material to avoid direct contact. Shipment should be done in the fastest way possible such as same day delivery or overnight courier service (26).

The harvest of the sperm from the epididymis should be carried out in room temperature in a clean environment, where instruments, as well as drapes and other equipment, are sterile. There are several techniques for epididymal sperm harvest.

One technique is the retrograde flushing technique (23,24,27,28), which uses the seminal plasma of another stallion (26) or semen extender (27). A syringe connected to a 14 to 18-gauge blunted needle catheter (27), a teat cannula or pipette tip (23) is inserted into the open end of the vas deferens and the seminal plasma or semen extender is flushed through the vas deferens and epididymal tail. In order to expel the spermatozoa, the tract is flushed with 10 – 20 ml of air or 5 – 10 ml of warm semen extender (26). Another approach is to make an incision in the junction of tail and body of epididymis were the semen extender can pass into a collection tube, so a second flushing is not needed (24).

Another method is the flotation method (1,27,29,30), in which 12 to 15 cuts are performed

along the tail of epididymis and vas deferens. A Petri dish with around 5 ml of warmed semen

extender is used to place the cut tissues inside. During a time span of 10 minutes the spermatozoa

swim out of the epididymis and vas deferens. The tissues can be removed afterwards and the

remaining semen extender containing the spermatozoa can be used for further processing. The

flotation method is an easy way of harvesting sperm; another similar method described by Dascanio

may give a larger quantity of spermatozoa (26). In this procedure first of the sperm of the vas

deferens is harvested separately by primarily removing the vas deferens from the tail of epididymis

and flushing it on its own into a pre-warmed centrifuge tube or similar container. The tail of

epididymis together with 5 – 10 ml warm semen extender, is put into a Petri dish and chopped into

small pieces two scalpel blades. The spermatozoa will be visible in small lumps of white color. For

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more spermatozoa to leave the chopped-up epididymis, it is left in the semen extender for 10 – 15 minutes. The mixture is afterwards filtered through sterile gauze sponge or a mesh filter used for semen collection into the same tube used for the collection of the spermatozoa of the vas deferens before. Warmed semen extender can be used to flush the petri dish and remaining material in the filter for the extraction of retained spermatozoa (26).

1.7 Andrological Examination

1.7.1 Morphological Examination of external Genitalia

As the whole penis needs to be viewed for an examination of the external genitalia the penis needs to be extracted from the sheath. As manual retraction is usually not tolerated, the best method is to tease the stallion or to perform the examination after semen collection (14,31). Alpha-2 sedatives should be avoided as it can cause paraphimosis. After the penis has been withdrawn from the sheath it can be examined entirely. The prepuce shall also be examined, also checking in between the skin folds. At the location where the urethra exits the gland penis it needs to be looked for smegma buildup also called beans. The penis can be cleaned with 40°C clean water and disposable paper towels. The penis and prepuce should be free from vesicular, proliferative or inflammatory lesions. Sarcoids, squamous cell carcinomas and equine coital exanthema may be found in this region.

The scrotum and its content are examined by palpation. The skin of the scrotum should be thin, smooth, elastic and freely movable from the testis and epididymis (31). Both testes are checked for symmetry, size, form and structure. To determine the position of the testis, the epididymis with its cranial head of epididymis and caudal tail of epididymis is palpated; it can eliminate the suspicion of testicular torsion. With the help of ultrasound or measuring tape the size and width of the testis can be established and used to calculate the testicular volume. The epididymis of both testes also need to be compared for size and symmetry. In case of deviations ultrasonography is also performed. A 5·0 MHz linear transducer is used for testicular ultrasound examinations but a 7·5 to 10·0 MHz transducer can be advantageous when details are viewed. All testicular parenchyma needs to be examined with the central vein and testicular artery.

Procedures such as viewing of internal accessory sex glands by ultrasound, color Doppler

ultrasound of scrotal contents or urethral endoscopy are not routinely performed but may be needed

to give additional information in search of a prognosis (31).

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1.7.2 Macroscopic semen evaluation

The gel-free semen fraction is evaluated for color and consistency. It should be white and opaque. Alterations may be of yellow color, which can suggest urine contamination or the presence of white blood cells; or pink color, which may indicate hemorrhage. The consistency is influenced by the semen concentration; it will be creamy if concentrated and more liquid when the semen concentration is lower (32). According to J. Crabtree (33) the pH value of normal stallion semen is between 7,2 to 7,7. The pH value can alternate according to the season, frequency of ejaculations and sperm concentration (14).

1.7.3 Microscopic semen evaluation

1.7.3.1 Subjective motility determination

The motility determination of a fresh semen sample is one of the most important investigations done to conclude fertility of the sample. All procedures should be conducted at +37°C, therefore all equipment touching the semen sample should be heated accordingly (33). The motility examination should be performed as shortly after the collection as possible (34).

The fresh semen sample is placed on a pre-warmed glass slide and viewed under the microscope, which preferably holds a heated stand for the glass slide to be placed on. Magnification is set to x10 or x40. After a general overview a preheated coverslip is placed on top of the drop of semen and the motility is evaluated in five different places. Fresh semen can have the tendency to clump or can be too concentrated for a good view, so a second investigation with a pre-warmed extender of a ratio 3:1 can be done (33). The examiner is focusing on the non-moving sperm and gives a broad estimate, if the sample is poor, fair, or good progressive motility (<30%, 30-59% and

≥ 60%) (34).

1.7.3.2 Computer-assisted sperm analysis

CASA terms “computer-aided sperm analysis” and “computer-assisted sperm analysis”. A

software is used to identify all spermatozoa viewed in a video, track their movement and calculate

the incurred data. To be able to conduct CASA, a microscope, video camera, video frame grabber

card, computer, software and a heated plate are needed. Nowadays there are multiple companies

marketing complete CASA systems (35). Spermatozoa are usually visualized with a dark

field/negative phase contrast/fluorescent optics microscope (36). The reliability of the examination

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with a CASA system is reliant on several factors such as the frame rate, settings and precision of the used devices, the adjustment of light and optic of the microscope, the temperature of the sample and the devices, counting chamber type and its depth, number of analyzed spermatozoa, sperm concentration, presence of debris in the sample, the used diluent, physiological status of the sperm, condition of the laboratory and executive personnel (35,37). Different authors suggest different set up adjustments for CASA depending on for example concentration and animal species. Good results depend very much on the users knowledge of CASA (36).

With help of the CASA system the motility assessment was improved by the objective measurement of the spermatozoal quality in movement. The CASA parameters have the advantage over direct visual classification, as it is objective and consistent. But for scoring hyperactivated motility of the spermatozoa it is still inferior to the subjective assessment. This problem is caused by the fact that the typical figure-of-8 pattern of movement in hyperactivation cannot be exactly apprehended, because the CASA system is working with a two-dimensional pattern movement.

This pattern is useful for evaluating the typical forward progressive movement of spermatozoa expressed in fresh semen (38).

1.7.3.3 Concentration and total sperm count

Sperm concentration can be determined using several methods, which are cell counting chamber, spectrophotometer, the NucleoCounter® or the flow cytometry (39,40). A CASA system can also be used to calculate the concentration of sperm, but it is said to not be very exact.

Total count of spermatozoa can be calculated with the volume of ejaculate and sperm concentration. The minimum requirement is set to be 100 million spermatozoa per milliliter for sperm concentration in the ejaculate in stallion. The fluctuation range can be from 100 till 350 million spermatozoa per milliliter (41).

The epididymis purpose is to store sperm cells for ejaculation (15). In a study conducted by Cary(1) 4485 billion sperm cells were harvest by retrograde flushing and 4886 billion sperm cells were obtained by flotation method from the epididymis of a stallion. The difference between left and right epididymal sperm volume are not significant, as Guimarães (28) had harvested 7758 ± 964,3 million sperm cells from the left and 8873 ± 1355 million from the right epididymis.

1.7.3.4 Morphology

The morphology of spermatozoa are usually evaluated with a bright-field microscope (100x

oil immersion), the sample is stained and air-dried as a smear. Stains such as eosin-nigrosin or India

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ink are generally used as background stains and Wright’s or Giemsa stains can also be used as cellular stains (14). Semen samples for wet-mount preparations are fixed in buffered formol saline and can be viewed by phase-contrast to evaluate morphology of sperm cells (42).

Morphological anormalies of sperm cells can be classified by different systems. Depending on the laboratory one may use one or another classification system, but in a single laboratory the same classification is used as a standard (43).

Cytoplasmic droplets are distinguished between proximal and distal cytoplasmic droplets.

They are seen as excess cytoplasma and are expelled from the spermatozoa, by moving from proximal to the distal end along the tail of spermatozoa during the transport inside the epididymis (42,44). In ejaculated samples cytoplasmic droplets are considered to be anomalies according to Brito (42), but according to Morrell (45) distal droplets are considered as physiological.

A maximum of 30% morphological anomalies are accepted in stallion semen (41). According to a study conducted by Varner (46) some sperm defects influence fertility more than others. Loose heads, head deformations, midpiece defects, undeveloped and premature sperm cells are unfavorable for fertility. Cytoplasmic droplets and bent tails are seen as less influential to a stallion’s sperm fertility. The amount of morphologically normal spermatozoa correlates positively with pregnancy rates (45).

1.7.3.5 Viability

Viability of sperm cells can be examined through a smear stained in a supravital dye. A combination of two dyes is usually used, were one dye penetrates damaged or compromised membranes and stains the dead sperm cell. The other dye is responsible for staining the background and making live sperm cells more visible. One of those stains is Eosin-Nigrosin Stain. Dead spermatozoa are stained in red as Eosin is penetrating the cell membrane and the living sperm cells are in white color with a background stained violet by Nigrosin (42,44).

1.7.3.6 Foreign Cell Count

With phase contrast microscope the foreign cell count can be determined. With the help of

size and shape the foreign material is distinguished from sperm cells. Foreign material in the semen

sample can be epithelial cells, premature germ cells, erythrocytes and leukocytes (41,47).

17

1.7.3.7 Agglutination

The adherence of Sperm cells among each other is referred to as Agglutination (47). Most usual the Sperm cells adhere together at their heads, the tail may still move freely. It is also possible an agglutination in star-shaped arrangements as well as clustered motionless sperm cells. The incidence of occurrence as well as type of agglutination are to be noted at the Semen examination (41,47).

1.8 Dilution and conservation of spermatozoa

1.8.1 Preparation of equine spermatozoa with fresh semen diluent

There are numerous advantages in adding semen extender to the semen sample. One of them is to increase the volume of the ejaculate. Due to the increase in volume, multiple females can be fertilized with one ejaculate. It also reduces the loss of sperm cells in the equipment and build-up of toxic byproducts of metabolism (48). Another objective of an extender is to provide effective antibiotic treatment when the semen contains pathogenic or potentially pathogenic organisms.

Antibiotics are often added to the extender by the manufacturer and can prevent the spread of venereal diseases or uterine infection (49,50). The survival rate of spermatozoa is also increased by adding an extender, making it important for shipping nationally or even internationally. Nowadays mares are bred more often with shipped semen. Therefore, the extender eliminates the stress and costs of shipping the mare or stallion to one another for breeding (48). Extenders also protect the spermatozoa. Unfavorable environmental conditions do not influence the sperm cells as easily as if they were dispersed in a protective fluid. Another advantage of the dilution with an extender is the proper evaluation of sperm motility, as the sperm cells can be viewed individually. The accuracy depends on clarity of the extender and quality of the microscope (51).

The dilution of equine sperm cells should be done with a high-quality diluent. The function of a compatible diluent for fresh equine spermatozoa is to provide energy, protect against changes in pH, temperature and osmotic pressure. In addition enzymatic systems in the Sperm cell need to be stabilized and membrane integrity sustained (52). The diluent has to maintain the spermatozoa motility and fertility. The diluent cannot interfere with the microscopic examination, so it has to be clear and free of debris (51).

The essential ingredients of fresh semen diluent are lipoproteins, metabolites and antibiotic

additives. Lipoproteins are mainly milk or egg yolk and responsible for membrane stabilization,

thus can protect the sperm cells from cold shock. Glucose, lactose and fructose can be used as

18

metabolites. To prevent bacterial growth antibiotics can be added as well. Egg yolk is a lipid additive, which is widely used for stallion spermatozoal extenders. Containing low-density lipoproteins, especially the phospholipid, in the egg yolk provides a protective action for stabilizing the spermatozoal membrane (53). Skim milk extenders can also be used to protect against cold shock. Most likely lipoproteins in milk function in similar manner as the egg yolk extender does for protection (51).

Spermatozoa are at risk of cold shock when rapidly cooling between 20°C and 5°C. During this range semen should be cooled slowly (54,55). Even more important is the reduction of seminal plasma in the sample to 5-20% if used for cooled storage. It improves the quality of semen by increased motility and fertility (53).

The pH of the diluent should be in the range of 6,2 to 7,2. To prevent a decrease in pH by accumulation of toxic metabolic waste products of spermatozoa a buffer can be added. The diluents osmolality should lay in between 300 and 400 mOsm per liter (52,54).

The dilution ratio is a minimum of 1:1 and maximum of 1:4 (56). Adding a diluent to the spermatozoa can increase their movement, but effects are usually first seen after ten to fifteen minutes after dilution (46).

1.8.2 Centrifugation

Centrifugation is mostly performed with diluted semen for 10-15 minutes at 400-600 x g.

During this procedure caution should be taken, as the improper centrifugation can result in spermatozoal damage. Depending on the semen sample the centrifugation should be customized, thus a sample with already great motility and velocity can tolerate greater centrifugation force. A considerable issue with centrifugation is the loss of roughly 25% of sperm cells in the supernatant.

Only if the insemination takes place directly after the procedure, the supernatant can be used. In

case of cooled-shipping or storage the supernatant needs to be discarded. Centrifugation with the

cushion technique has made it possible to use greater force without damaging the spermatozoa. It is

a solution which is placed below the diluted semen to prevent compacting of spermatozoa at the

bottom of the tube during centrifugation. Centrifugation at 1000 – g for 20 min is made possible

with this method without causing major destruction and allowing a recovery rate of 90% and more

(53).

19

1.8.3 Cooling of fresh stallion semen

Fresh semen stored at 37 °C would deteriorate rapidly. Most raw ejaculates will have no motile sperm after only 3-4 hours at 37 °C and 5-6 hours at 20-24 °C. A transition in sperm membranes from liquid crystalline to a gel state is induced when cooling the sample to 5 °C. At body temperature the metabolism of spermatozoa is maximal, at room temperature already decreased. Permanent cellular damage, such as peroxidation of membrane lipids result in membrane damage and cells wearing out, due to waste products building up and increasing acidity of semen.

For each 10 °C of decreased temperature, the cellular metabolism is reduced by 50%. Due to this, the semen stored at 5 °C has only a metabolic need of 10% of what it would need at 37 °C.

Consequently, the spermatozoa is also not producing as many waste products, lipid peroxidation occurs more slowly and the cells do not wear out as fast. However, cooling is also a stress factor and can also cause cellular damage. The cell structure may be affected directly, by for example rupturing of the cell membrane, or indirectly by altering cellular functions, such as slowing down the metabolic processes (54).

Cooling stallion spermatozoa rapidly from room temperature to 5 °C causes partially irreversible damage such as abnormal swimming pattern, rapid loss of motility, acrosome damage, plasma membrane damage, reduced metabolism and loss of intracellular components. These damages are collectively referred to as cold shock. Indirect cooling damages may first be evident after several hours after cooling and re-warming. Damages can result due to changes that occur as the plasma membrane transitions from liquid-crystalline to a gel state. Additives in the extenders can minimize this damage but careful slow cooling during the transition period is also important (54).

1.8.4 Cryopreservation of stallion semen

Cryopreservation is the storage of spermatozoa at temperatures below 0 °C. Frozen semen is

most commonly stored in liquid nitrogen with a temperature of -196 °C, were the metabolism of

Spermatozoa is stopped. The sperm cells do not only need to survive the storage of 196 °C but also

the critical phases of cooling, freezing and thawing. Critical temperatures are mentioned as +19 °C

till +9 °C (57) and -15°C till -60 °C (58,59). The freezing point for extra- and intracellular fluids

range from 0 °C till -5 °C. From -5 °C till -15 °C ice crystals are formed. With the increasing of

crystallization the osmotic pressure rises in the remaining non-frozen fluid (52,59). Water escapes

the sperm cells and the cells dehydrate. Large ice crystals are forming in the extracellular fluid

when the cooling rate is slow. The Sperm cells are remaining in the spaces in between the crystals,

20

the forming of crystals inside the sperm cells is prevented by the dehydration (59). The perfect speed of freezing is the golden middle solution. It has to be fast enough to not dehydrate the sperm cells to much but also slow enough to prevent intracellular crystals from forming (58,60,61). The optimal rate of cooling without adding cryoprotectants is -29 °C per minute, when a cryoprotectant is added -60 °C per minute can be used as cooling rate (61). As soon as the Spermatozoa have reached a temperature below -60 °C they are inactive and in a stabile state. These samples can be stored for many years (62,63).

2. RESEARCH METHODS AND MATERIALS

This research work was carried out at the Large Animal Clinics Animal Reproduction Laboratory of the Lithuanian University of Health Sciences (LUHS) in Kaunas, Lithuania. The sampling procedure and examination procedures were undertaken between October 2018 and June 2019.

For this study, eight horses of Arabian, English Thoroughbred, Holstein, Frisian and Westphalian breed were subject to castration for other reason than this study. Samples from the epididymis of the castrated testes were taken in the Large Animal Clinic. The age ranged from four to ten years and weight ranged from 430 to 600 kg.

Information about the horses was obtained at the Large Animal Clinic from the veterinarians about horse age, breed, weight, reason of castration, prior reproduction of the stallion, systemic drugs used for anesthesia, as well as local drugs used in the testes and the horse’s health status.

Two labeled sample tubes, one with 10.0 ml pre-warmed Ringer B. Braun solution (B. Braun

Melsungen AG, Germany) and one with 10.0 ml EquiPlus extender (Minitube, GmbH, Tiefenbach,

Germany). For EquiPlus, 0.67g extender powder were solved into pre-warmed 10 ml distilled

water. Extenders were prepared beforehand and warmed to a temperature of 37 ± 0.5 °C. Samples

were taken as soon as possible with a maximum of one hour after castration. With a scalpel the

Cauda epididymis was incised, trying to avoid large blood vessels to minimize blood

contamination. The sperm was collected with a pipette from the incision site and transferred into the

two sample tubes. The amount of sperm collected was the same for both tubes as measured with the

pipette. For the sample collection of the Corpus epididymis and Caput epididymis the sites were

incised with a scalpel and a compression sample was taken with microscopic slides. Inside an

isolating box for fresh semen transportation the samples were brought to the Animal Reproduction

Laboratory, which is located in a 2 minutes walking distance from the Large Animal Clinic. In one

hour samples were used for primarily (0 hours) sperm pH, subjective motility, motility SCA,

viability, concentration and morphology examination. After primary semen quality evaluation every

21

sample was divided in two parts (each tube 5 ml aliquots). One part of samples (Group 1 – Ringer

20 °C and EquiPlus 20 °C) were incubated at 20

C ± 2

C in the acclimatized box – „room

temperature“ (Friocell, Germany) and the second part of samples (Group 2 – Ringer 4 °C and

EquiPlus 4 °C) were kept at 4

C ± 2

C in a refrigerator for 72 hours. Sperm viability, pH,

subjective motility and objective motility SCA were checked 0 hour and after 24, 48, and 72 hours

of incubation (day 1, day 2, day 3 and day 4). In total, 140 tests for sperm viability, motility,

motility SCA and pH were performed from eight horse’s ejaculates.

22

Analysis:

• Motility

• Viability

• pH

• Concentration Analysis:

• Morphology

Motility, Viability & pH Analysis after:

• 24 hours

• 48 hours

• 72 hours Motility, Viability & pH

Analysis after:

• 24 hours

• 48 hours

• 72 hours

Motility, Viability & pH Analysis after:

• 24 hours

• 48 hours

• 72 hours

Motility, Viability & pH Analysis after:

• 24 hours

• 48 hours

• 72 hours Analysis:

• Motility

• Viability

• pH

• Concentration Caput and Corpus

Epididymis Sample collection by compression

Analysis:

• Morphology

Fig. 2: Research construction

Testes

Transport to laboratory within 1 hour

Cauda Epididymis Sample collection

by pipetting Castration

EquiPlus Solution

Ringer Solution

Storage at

4 °C Storage at

20 °C Storage at

4 °C Storage at

20 °C Information collection of horse:

• Age

• Breed

• Weight

• Health status

• Reason for castration

• Anesthesia drugs

23

2.1 Motility assessment

Subjective motility as well as Computer assisted sperm analysis (Sperm Class Analyzer (SCA, Microptic, Spain)) was performed under a phase-contrast microscope Olympus BH2 with prewarmed 37 ± 0.5 °C stage (Nicon ECLIPSE 50i, Japan). The equine sperm motility assessment was performed with 37 ± 0.5 °C at maximum activity, the temperature was reached by placing the samples in a water bath (Memmert, Germany) for 5 minutes. Additionally, equipment, which is in near contact with the sample, such as microscopic slides and pipette tips, are pre-warmed. A drop of warm sperm (5 – 10 µl) is placed on a 37 °C glass slide and covered with a warm lid.

For subjective motility the sperm motility is measured by eye to determine the percentage of progressive spermatozoa in three to five fields of the microscopic view. The end result is calculated by the arithmetic mean of all measurements.

Objective motility was determined with the same microscope with help of a chamber. The microscopic image is converted into a digital image on a computer with a computerized sperm motility evaluation system SCA (MICROPTIC S.L., Spain) installed. Each sample was evaluated in at least three fields of vision and the computer recorded the total percent sperm motility (Motility SCA) and percent of progressive motility (Progressive motility)in the program.

2.2 Viability assessment

Viability was examined in dry preparations with Eosin/Nigrosin dye. Onto a glass slide one

drop (5 – 10 µl) of sperm sample as well as one drop (5 – 10 µl) of dye are placed and mixed

together. The mixture is spread across the slide thinly and let air-dry. The sample was viewed under

a phase contrast microscope (Olympus BH2, Olympus Optical Co., Ltd., Japan) under 1000x

magnification with immersion and a total of 200 sperm cells were counted. It was distinguished

between alive and dead cells and recorded (Fig. 3).

24

Fig. 3: Stained Spermatozoa for Viability, microscopic view.

2.3 PH evaluation

Upon arrival in the Animal Reproduction Laboratory the samples were warmed in a 37 ± 0.5

°C water bath prior to any assessment for 5 minutes. Afterwards the samples were measured for pH

with an electronic pH meter (AB 150, Fisher Scientific Accumet®(Fig.4)). The standard H ion

electrode was used to measure the H ion concentration in the diluted equine epididymal sperm

samples. The electrode was inserted into the sample tube and the pH value shown on the devises

screen was documented.

25

Fig. 4: Measurement of pH with AB 150, Fisher Scientific Accumet®

2.4 Sperm concentration

The sperm concentration was measured in a hematocytometric camera; Neubauer cell counting chamber. The sperm samples were diluted with distilled water at a 1:20 ratio and mixed thoroughly. Subsequently the Neubauer cell counting chamber was filled and the sperm count was calculated by counting sperm cells in five large squares (80 small squares) or 0.2 mm

. The sperm count was performed three times in three different squares and the mean value of the three countings was calculated. The sperm concentration is then determined by the subsequent formula:

K = S × P × 5 × 10 × 1000 where:

K is the concentration of the sperm sample

S is the amount of sperm cells counted in 80 small squares

P is the dilution ratio, the coefficient is 5 on account of that sperm were counted in a 0.2 mm

chamber area, due to the fact that the chamber depth is 0.1 mm the multiplier is 10 and 1000 is used to multiply to convert to 1 ml.

2.5 Morphological evaluation

The evaluation of sperm head pathologies and sperm tail pathologies were conducted separately.

Morphological tail pathologies were examined in wet preparations by Hancock method. The

sperm was diluted in buffer formalin solution at a ratio of 1:10. One drop of diluted sperm sample

26

(5 – 10 µl) was placed on a slide and covered by a cover slide. The preparation is let to settle to the bottom of the slide for several minutes before evaluating it with a phase contrast microscope (Olympus BH2, Olympus Optical Co., Ltd., Japan) with a magnification of 400-fold. 200 sperm cells were counted in each sample. Pathologies, which were documented, are normal loose heads without tail, acrosome defects, acrosome abnormalities and vacuoles. Also, cervical lesions such as abnormal mid-pieces, proximal and distal cytoplasmic droplets are recorded as well as tail abnormalities, which are simple bent tail, coiled tail and double coiled tail.

Morphological head pathologies (pear shape, narrow at base, abnormal contour, undeveloped, loose abnormal head, narrow, big, little, normal, short broad) were examined in dry preparations stained with methylene blue dyes (SpermBlue, MICROPTIC S.L., Spain). One drop of sperm sample (5 – 10 µl) is placed on a glass slide together with one drop (5 – 10 µl) of dye and mixed together. The mixture is afterwards spread thinly across the glass slide and let to air-dry. The sample is viewed under a phase contrast microscope (Olympus BH2, Olympus Optical Co., Ltd., Japan) under 1000x magnification with immersion. In each sample 500 sperm cells are counted from different areas of the smear and evaluated for sperm head pathologies. Such head pathologies are pear-shaped, narrow-base, abnormal contour, undeveloped, loose abnormal heads, narrow- headed, big heads, small heads, short and wide heads as well as paracentrical tail attachments to the mid-piece. Total percentage of pathologies from tails and total percentage of other pathologies as well as total percentage of all pathologies (heads, tails and others) were calculated.

2.6 Statistical analysis

Statistical analysis was performed using Excel and IBM SPSS statistics 20 for Windows (SPSS

for Windows 9.0, SPSS Inc., Chicago, IL, USA). The data included in the model were analyzed

using descriptive statistics (mean ± SD) and 1-way ANOVA analysis. The differences between the

investigated groups were analyzed by the LSD method (α = 5%). The differences were considered

to be statistically significant when: * P < 0.05; ** P < 0.01; and *** P < 0.001.

27

3. RESEARCH RESULTS

3.1 Descriptive statistics 3.1.1 Motility in two different diluents

The mean sperm motility was measured in two different diluents, one in Equiplus Extender and one in Ringer B. Braun Solution. Each sample was subjectively measured, as well as by SCA and for Progressive motility. Mean subjective motility was measured with 29.06 ± 1.95% in Equiplus solution and 27.69 ± 1.87% in Ringer solution, having a mean difference of 1.37%

(p>0.05). When measured with SCA the highest mean motility was measured with 67.23 ± 2.24%

in Equiplus and 64.39 ± 2.27% in Ringer solution, with a mean difference of 2.84% (p>0.05).

Meanwhile, if only the progressive motility is measured in Equiplus solution, it is 24.65 ± 1.78%

and the lowest mean motility is measured in Ringer solution with 23.45 ± 1.71%, a mean difference of 1.2% (p>0.05) (Fig. 5).

Fig. 5: Mean motility values measured subjectively, by SCA and SCA progressive motility comparing Equiplus and Ringer solution

3.1.2 Viability and pH in two different diluents

The mean viability was measured at 83.3 ± 1.1% in Equiplus solution and 78.5 ± 1.59% in Ringer solution. The mean difference of 4.8% is significant (p<0.05). The mean pH in the Equiplus solution was higher with a value of 6.64 ± 0.03, in Ringer solution a mean pH value of 6.35 ± 0.03 was measured. The mean difference of 4.37% is not significant (p>0.05) (Fig.6).

0 10 20 30 40 50 60 70 80

Subjective Motility SCA Motility Progressive Motility

Percent, %

Equiplus Ringer

28

Fig. 6: Mean viability and pH of sperm in Equiplus and Ringer solution

3.1.3 Concentration in 2 different diluents

The mean sperm concentration in Equiplus was measured to be 220.8±9.82 sperm/ml, in Ringer a concentration of 233.7±9.53 sperm/ml. The difference of 5.52% in the two different diluents was not significant (p>0.05).

3.1.4 Motility at two different temperatures

A mean difference of 4.68% (p>0.05) for subjective motility at 20 °C and 4 °C was measured.

A mean difference of 9.25% (p>0.05) for SCA measured motility and progressive motility mean difference of 6.6% (p<0.05) was measured between 20 °C and 4 °C mean values. The subjective motility, SCA motility and progressive motility differed significantly from each other (p<0.01) (Fig. 7).

6,30 6,35 6,40 6,45 6,50 6,55 6,60 6,65 6,70

76 77 78 79 80 81 82 83 84

Equiplus Ringer

pH

Percent, %

viability ph

29

Fig. 7: Mean motility according to assessment method in 20 °C and 4 °C

3.1.5 Viability and pH at two different temperatures

For viability a mean difference of 2.14% (p>0.05) was recorded at 20 °C and 4 °C. The mean difference in pH between 20 °C and 4 °C was measured to be 2.17% (p<0.01) (Fig. 8).

Fig. 8: Mean viability and pH value at 20 °C and 4 °C

0 10 20 30 40 50 60 70

20°C 4°C

Percent, %

Subjective Motility SCA Motility Progressive Motility

6,34 6,36 6,38 6,4 6,42 6,44 6,46 6,48 6,5 6,52

77 78 78 79 79 80 80 81 81

20 °C 4 °C

pH

Percent, %

Temperature

Viability pH

30

3.2 Sperm quality values at 24, 48 and 72 hours

Subjective motility mean value at 0 hours is 27.19% (p<0.001) higher than after 24 hours.

From 24 to 48 hours the mean difference was decreasing the value by 18.89% (p<0.001). A decrease of 6.03% (p<0.001) was measured from 48 to 72 hours. Motility measured by SCA on 0 hours was higher by 12.72% (p<0.05) compared to 24 hours. From 24 hours to 48 hours a decrease of 21.91% (p<0.001) was recorded. A decrease from 48 to 72 hours measured by SCA was 19.18%

(p<0.001). Progressive motility is highest at 0 hours, with a mean difference compared to 24 hours of 30.27% (p<0.001). A decrease from 24 to 48 hours by 13.77% (p<0.001) is measured and another decrease from 48 to 72 hours by 7.63% (p<0.001), making values at 72 hours the lowest (Fig. 9).

Fig. 9: Sperm motility after 0, 24, 48 and 72 hours according to assessment method 3.2.1 Subjective motility in different diluents

Mean motility value at 0 hours in Equiplus solution at 20 °C storage temperature decreased from 0 to 24 hours by 21.89% (p<0.001), from 24 to 48 hours by 21.11% (p<0.001) and from 48 to 72 hours by 14.56% (p<0.05). In Equiplus solution at 4 °C storage from 0 to 24 hours is a decrease of 24.67% (p<0.001) marked. From a storage time of 24 to 48 hours the mean subjective motility value decreases by 16.67% (p<0.005). From 48 to 72 hours an increase by 1.67% (p>0.05) was recorded. In Ringer solution at a storage temperature of 20 °C a decrease of 32.78% (p<0.001) from 0 to 24 hours was measured, from 24 to 48 hours a 18.33% (p<0.001) decrease and from 48 to 72 hours a decrease of 6.0% (p>0.05) was recorded. In Ringer solution with a storage temperature of 4

0 10 20 30 40 50 60 70 80 90 100

0 24 48 72

Percent, %

Hours

subjective SCA progressive

31

°C a decrease of 29.44% (p<0.001) from 0 to 24 hours was noted as well as a descending value from 24 to 48 hours by 19.45% (p<0.001) and from 48 to 72 hours by 5,22% (p>0.05) (Fig. 10).

Fig. 10: Mean subjective motility on 0, 24, 48, 72 hours according to Equiplus solution at 20 °C, Equiplus solution at 4 °C, Ringer solution at 20 °C and Ringer solution at 4 °C.

3.2.2 SCA motility in different diluents

Mean motility value measured by SCA at 0 hours in Equiplus solution at 20 °C storage temperature decreased from 0 to 24 hours by 8.88% (p<0.001), from 24 to 48 hours by 15.64%

(p>0.05) and from 48 to 72 hours by 28.82% (p<0.001). In Equiplus solution at 4 °C storage from 0 to 24 hours is a decrease of 6.5% (p>0.05) marked. From a storage time of 24 to 48 hours the mean subjective motility value decreases by 12.48% (p>0.05). From 48 to 72 hours an increase by 9.86%

(p>0.05) was recorded. In Ringer solution at a storage temperature of 20 °C a decrease of 22.84%

(p<0.001) from 0 to 24 hours was measured, from 24 to 48 hours a 30.38% (p<0.001) decrease and from 48 to 72 hours a decrease of 14.05% (p<0.05) was recorded. In Ringer solution with a storage temperature of 4 °C a decrease of 12.68% (p>0.05) from 0 to 24 hours was noted as well as a descending value from 24 to 48 hours by 29.12% (p<0.001) and from 48 to 72 hours by 23.1%

(p<0.001) (Fig. 11).

0 10 20 30 40 50 60 70

0 24 48 72

Percent, %

Hours

Equiplus 20°C Equiplus 4°C Ringer 20°C Ringer 4°C

32

Fig. 11: Mean SCA motility on 0, 24, 48, 72 hours according to Equiplus solution at 20 °C, Equiplus solution at 4 °C, Ringer solution at 20 °C and Ringer solution at 4 °C

3.2.3 Progressive motility

Mean progressive motility value measured at 0 hours in Equiplus solution at 20 °C storage temperature decreased from 0 to 24 hours by 28.02% (p<0.001), from 24 to 48 hours by 13.79%

(p<0.01) and from 48 to 72 hours by 11.96% (p<0.01). In Equiplus solution at 4 °C storage from 0 to 24 hours is a decrease of 22.41% (p<0.001) marked. From a storage time of 24 to 48 hours the mean subjective motility value decreases by 12.77% (p<0.05). From 48 to 72 hours a decrease by 4.72% (p>0.05) was recorded. In Ringer solution at a storage temperature of 20 °C a decrease of 38.35% (p<0.001) from 0 to 24 hours was measured, from 24 to 48 hours a 13.1% (p<0.05) decrease and from 48 to 72 hours a decrease of 6.97% (p>0.05) was recorded. In Ringer solution with a storage temperature of 4 °C a decrease of 32.33% (p<0.001) from 0 to 24 hours was noted as well as a descending value from 24 to 48 hours by 15.43% (p<0.01) and from 48 to 72 hours by 6.86% (p>0.05) (Fig. 12).

0 10 20 30 40 50 60 70 80 90 100

0 24 48 72

Percent, %

Hours

Equiplus 20 °C Ringer 20 °C Equiplus 4 °C Ringer 4 °C

33

Fig. 12: Mean progressive motility on 0, 24, 48, 72 hours according to Equiplus solution at 20 °C, Equiplus solution at 4 °C, Ringer solution at 20 °C and Ringer solution at 4 °C

3.2.4 Viability

The mean viability of the sperm samples is decreasing by progressing time. Until 24 hours a decrease of 4.73% (p>0.05), after 48 hours a decrease from 24 hours is noted by 4.81% (p<0.05), after another 24 hours the viability has decreased by 6.74% (p<0.01) to its lowest value (Fig. 13).

Fig. 13: Mean viability of sperm according to time

Mean viability value measured at 0 hours in Equiplus solution at 20 °C storage temperature decreased from 0 to 24 hours by 1.55% (p>0.05), from 24 to 48 hours by 6.0% (p>0.05) and from 48 to 72 hours by 4.85% (p>0.05). In Equiplus solution at 4 °C storage from 0 to 24 hours is a

0 10 20 30 40 50 60 70

0 24 48 72

Percent, %

Hours

Equiplus 20 °C Ringer 20 °C Equiplus 4 °C Ringer 4 °C

0 10 20 30 40 50 60 70 80 90 100

0 24 48 72

Percent, %

Hours

34

decrease of 1.2% (p>0.05) marked. From a storage time of 24 to 48 hours the mean viability value decreases by 4.05% (p>0.05). From 48 to 72 hours a decrease by 2.85% (p>0.05) was recorded. In Ringer solution at a storage temperature of 20 °C a decrease of 8.6% (p>0.05) from 0 to 24 hours was measured, from 24 to 48 hours a 4.8% (p>0.05) decrease and from 48 to 72 hours a decrease of 10.6% (p>0.05) was recorded. In Ringer solution with a storage temperature of 4 °C a decrease of 7.55% (p>0.05) from 0 to 24 hours was noted as well as a descending value from 24 to 48 hours by 4.4% (p>0.05) and from 48 to 72 hours by 8.65% (p>0.05) (Fig. 14).

Fig. 14: Mean viability according to time compared to different solutions and temperature

3.2.5 pH values

A mean difference in pH from 0 to 24 hours can be measured by 4.52% (p<0.001), another decrease by 2.62% (p<0.01) from 24 to 48 hours is noted and the lowest pH measured at 72 hours with a decreased mean compared to 48 hours by 13.66% (p>0.05) is recorded (Fig. 15).

60 65 70 75 80 85 90 95

0 24 48 72

Percent, %

Hours

Equiplus 20°C Ringer 20°C Equiplus 4°C Ringer 4°C

35

Fig. 15: Mean pH value on 0, 24, 48 and 72 hours

3.2.6 pH of 2 diluents, at 4 and 20°C on 3 days

Mean pH value measured at 0 hours in Equiplus solution at 20 °C storage temperature decreased from 0 to 24 hours by 3.66% (p<0.01), from 24 to 48 hours by 4.47% (p<0.001) and from 48 to 72 hours by 3.72% (p<0.01). In Equiplus solution at 4 °C storage from 0 to 24 hours is a decrease of 2.51% (p<0.01) marked. From a storage time of 24 to 48 hours the mean subjective motility value decreases by 2.49% (p<0.05). From 48 to 72 hours a decrease by 1.63% (p>0.05) was recorded. In Ringer solution at a storage temperature of 20 °C a decrease of 6.53% (p<0.001) from 0 to 24 hours was measured, from 24 to 48 hours a 15.61% (p>0.05) decrease and from 48 to 72 hours a decrease of 0.32% (p>0.05) was recorded. In Ringer solution with a storage temperature of 4 °C a decrease of 5.52% (p<0.001) from 0 to 24 hours was noted as well as a descending value from 24 to 48 hours by 1.8% (p>0.05) and from 48 to 72 hours by 0.3% (p>0.05) (Fig. 16).

6,0 6,1 6,2 6,3 6,4 6,5 6,6 6,7 6,8 6,9 7,0

0 24 48 72

pH

hours