LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY
Faculty of Veterinary Medicine
Niels Henry Edgar Olsén
ANALYSIS OF CRYOPRESERVED DAIRY AND BEEF BULL
SEMEN QUALITY AFTER THAWING
KRIOKONSERVUOTOS PIENINIŲ IR MĖSINIŲ VEISLIŲ
BULIŲ SPERMOS KOKYBĖS TYRIMAI PO ATŠILDYMO
MASTER THESIS
of Integrated Studies of Veterinary Medicine
Supervisor: Dr. Neringa Sutkevičienė
2 THE WORK WAS DONE IN THE DEPARTMENT OF LARGE ANIMAL CLINIC
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3 TABLE OF CONTENTS SANTRAUKA ...5 SUMMARY ...6 INTRODUCTION ...7 LITERATURE REVIEW ...8
1.1 Cattle bull reproductive tract ... 8
1.2 Morphology of cattle semen ... 8
1.2.1 The sperm head ... 8
1.2.2 The sperm tail ... 9
1.3 Cattle spermatogenesis ... 9
1.4 Fertility of frozen and fresh semen ... 9
1.5 Thawing effect on quality ... 10
1.6 Breed standards ... 10
1.7 Heritable measurements ... 10
1.8 Method of semen collection ... 11
1.9 Andrological Examination ... 11
1.9.1 Morphological examination of external genitalia ... 11
1.9.2 Macroscopic semen evaluation ... 12
1.9.3 Microscopic semen evaluation ... 12
1.10 Insemination techniques ... 14
1.11 Semen Storage ... 14
1.11.1 Frozen semen... 15
1.11.2 Semen extenders ... 15
RESEARCH METHODS AND MATERIALS ...16
2.1 Motility assessment ... 16
4
2.3 Sperm concentration ... 17
2.4 Morphological evaluation ... 18
2.5 Statistical analysis ... 19
RESEARCH RESULTS ...20
3.1 Sperm quality values and descriptive results at 0 hours and after 5 hours ... 20
3.1.1 Motility between two cattle types ... 20
3.1.1 Viability between two different cattle types ... 23
3.1.2 Concentration between two different cattle types ... 24
3.1.3 Morphology of different cattle types ... 25
3.1.4 Fertility rate between two different cattle types ... 29
DISCUSSION OF RESULTS ...30
CONCLUSIONS ...32
RECOMMENDATION ...33
ACKNOWLEDGEMENT ...34
5 KRIOKONSERVUOTOS PIENINIŲ IR MÈSINIŲ VEISLIŲ BULIŲ SPERMOS
KOKYBĖS TYRIMAI PO ATŠILDYMO
Niels Henry Edgar Olsén
Magistro baigiamasis darbas
SANTRAUKA
Mūsų darbo tikslas buvo išanalizuoti ir palyginti pieninių ir mėsinių veislių bulių spermatozoidų judrumą, gyvybingumą, morfologinius pokyčius ir apvaisinimo procentą po spermos dozės atšildymo. Spermos tyrimai atlikti Lietuvos sveikatos mokslų universiteto (LSMU) Stambiųjų gyvūnų klinikos Gyvūnų reprodukcijos laboratorijoje.
Iš viso buvo ištirta 23 bulių (14 pieninių ir 9 mėsinių veislių) šaldytos spermos mėginai. Spermos mėginiai iki tyrimo buvo laikomi diuaro inde skystame azote. Spermos šiaudeliai buvo atšildomi 37,0°C temperatūros vandens vonioje. Kiekvieno mėginio spermos kokybė buvo nustatoma iš karto po atšildymo ir po penkių valandų. Progresyvus ir bendras spermatozoidų judrumas buvo nustatomas subjektyviai ir kompiuterine spermos jurumo analizės programa SCA. Pieninių veislių bulių spermatozoidų judrumas iš karto po atšildymo nustatytas didesnis tiriant visomis trimis skirtingomis tyrimo technikomis (56,79±4,88 proc. subjektyvus, 85,44±3,8 proc. SCA, 60,85±5,69 proc. progresyvus) (p>0.05). Po 5 valandų subjektyvus ir progresyvus spermatozoidų judrumas taip pat didesnis (34,57±4,08 proc., 60,85±5,69 proc.) nustatytas pieninių veislių bulių mėginiuose. SCA judrumas po 5 valandų didesnis buvo mėsinių veislių bulių mėginiuose (66,38 ± 4,54 proc.). Pieninių veislių bulių spermatozoidų gyvybingumas nustatytas didesnis tiek iš karto po atšildymo (58,5±2,26 proc.), tiek ir po penkių valandų (34,36±1,97 proc.) (p>0,01). Spermatozoidų koncentracija dozėje didesnė buvo pieninių veislių bulių ir siekė 21,01±1,29 mln. Spermatozoidų galvučių patologijų daugiau buvo nustatyta mėsinių veislių bulių spermoje (1,47±2,38 proc.), nei pieninių veislių bulių spermoje (p>0.05). Spermatozoidų uodegėlių, o taip pat ir kitų patologijų kiekis buvo rastas didesnis mėsinių (1,92±2,04 proc.) nei pieninių veislių bulių spermoje (p>0.05). Pieninių veislių bulių spermos apvaisinimo procentas buvo 4,42±4,32 proc. didesnis nei mėsinių bulių ir siekė 48,6±1,94 proc. (p<0.05).
Raktažodžiai: Galvijai, pieniniai, mėsiniai, spermatozoidai, atšildymas, judrumas, gyvybingumas, morfologija
6 ANALYSIS OF CRYOPRESERVED DAIRY AND BEEF BULL SEMEN QUALITY
AFTER THAWING
Niels Henry Edgar Olsén
Master Thesis
SUMMARY
The objective of this study was to analyze and compare sperm motility, viability, morphologies, and fertility between beef and dairy bulls after thawing of the semen dose. The analysis of the sperm was performed in the Animal Reproduction Laboratory of Large Animal Clinic at the Lithuanian University of Health Science (LUHS).
Cryopreserved semen samples of 23 cattle (14 of dairy breeds and 9 of beef breeds) were analyzed. The samples were stored in a liquid nitrogen tank prior to analysis. The semen straws were thawed in a 37.0°C water bath. The analysis of the sperm quality of each sample was performed both directly after thawing and after five hours. Progressive as well as total sperm motility were both measured subjectively and via SCA computer assisted program. Dairy bulls showed higher motility rates with all three different measuring techniques directly after thawing (56.79 ± 4.88% subjectively, 85.44 ± 3.8% SCA, 60.85 ± 5.69% progressive) (p>0.05). After five hours, the subjective as well as the progressive motility were also higher (34.57 ± 4.08%, 60.85 ± 5.69%) in dairy bulls. SCA motility was measured higher in beef bulls after five hours (66.38 ± 4.54%). Sperm viability value measured was higher in dairy directly after thawing (58.5 ± 2.26%) as well as after five hours (34.36 ± 1.97%) (p>0.01). Dairy bulls had the highest measured value for concentration/dose at 21.01 ± 1.29 mil. Pathologies in the head were 1.47 ± 2.38% more common in beef than dairy bull sperm (p>0.05). There were also more tail and other pathologies found in beef than in dairy bull sperm (1.92 ± 2.04%) (p>0.05). Dairy bull sperm showed higher fertility rate than the beef bulls (48.6 ± 1.94%) with a mean difference of 4.42 ± 4.32% (p<0.05).
7
INTRODUCTION
The qualities of a bull are significant no matter the size of the farm nor if it is a bull raised for beef cattle or dairy cattle. Therefore, when the option and availability arise to choose a new bull, you need to select one that fits your qualifications and expectations. Artificial insemination has become the most common method used for insemination in all over Europe (1-3).The qualities of the semen and its fertility rate can make or break a lineage of a bull and it is therefore important to know the bulls breeding soundness evaluation (BSE) before purchase or insemination. In a study by Thibier et al. (2002), artificial insemination had been used in one-fifth of breedable females in the study’s responding countries and most of it was deep-frozen (4).
It is important to know that cryopreservation can have a permanent effect on the semen quality as well as alter the structure and function of the sperm (5), and that the quality that can be evaluated before preservation (fresh) will not be the same as after cryopreservation (6). Even though cryopreserved semen has a long lifespan, fresh semen still has an improved reproductive performance (7).
Beef and dairy bulls and their semen are similar in many ways, but comparing them can show if they are equal in semen evaluation as well. A comparison can also provide necessary information regarding if one type of bull has been improved upon more than the other, either due to higher demand in the world for that type of bull, or simply via evolution and manual selection of the highest fertility bull creating a gap between the two types in the evaluation.
The objective of this study was to research and analyze difference in quality between dairy and beef bull sperm after thawing, as well as their fertility.
Tasks of the work:
1. To analyze and compare beef and dairy bull sperm motility directly after thawing, and after five hours.
2. To analyze and compare beef and dairy bull sperm viability directly after thawing, and after five hours.
3. To analyze and compare beef and dairy bull sperm concentration after thawing. 4. To analyze and compare beef and dairy bull sperm morphology after thawing. 5. To analyze and compare beef and dairy bull sperm fertility.
8
LITERATURE REVIEW
1.1 Cattle bull reproductive tract
The bull reproductive tract has multiple organs and systems that work in synergy for the production, transport, and transfer of spermatozoa, just like the norm is with other male mammary reproductive systems. The reproductive system can be divided functionally into the previously mentioned parts: production, transport, transfer. The production of spermatozoa occurs inside the paired testes that lie in the scrotum, connected with the spermatic cord, leading to the second part: transport. Transport is done via, to name a few, a system of tubules, ductus deferens, and urethra to allow the spermatozoa to mature, storage, as well as fluid for eased movement. Transfer of spermatozoa from the bull to the cow is accomplished via the erected penis as well as ejaculation of said spermatozoa, either via intromission, or collection of the ejaculate and artificial insemination. The bull reproductive tract also includes accessory glands that provide the reproductive system with its own importance such as nutrients and protectants (8).
1.2 Morphology of cattle semen
The bull spermatozoon is similar to those of dogs, cats, humans, horses, or other male mammalian spermatozoa, consisting of a flat spatulate head, a midpiece, and a tail (with principal and end pieces), where the heads purpose is to lead the way for gamete fusion with the aid of flagellar motility from the tail. The head only contains axonemal microtubules, while both the head and flagellum are both enclosed tightly by the plasma membranes and also contains a small amount of cytoplasm inside (9,10).
1.2.1 The sperm head
The sperm head is of simple structure and can be summarized as a specialized nucleus package, containing condensed chromatin, DNA, surrounded by a simple nuclear membrane as well as an acrosome on the anterior aspect. The head contains two main enzymes, acrosin, and hyalurenodase (11). The acrosome plays a crucial prerequisite role for fertilization – the site of capacitation and acrosome reaction - so it needs to be intact. It contains hydrolytic enzymes and molecules needed for fertilization, and is surrounded by a membrane (10).The sperm fuses with the oocyte at the equatorial region and the anterior border of the postacrosomal region and postnuclear cap (9).
9 1.2.2 The sperm tail
As in most mammalian species the flagellum consists of four distinct segments. It consists of the connecting piece (the neck), the middle piece (midpiece), the principal piece, and the endpiece, connecting the annulus to the flagellum. The sperm tail is made up of a central axoneme, a mitochondrial sheath, outer dense fibers, and fibrous sheath. These parts are the main structural components of the sperm tail.
The motility of the sperm is provided by a coordination of waves from the flagellar bending, progressing from the neck down until the end of the tail. The bending is a result from forces generated between the peripheral central doublets of the axoneme. Each axoneme on each side of the center works in opposition to the other creating an alternating beat of contractions, creating motility (9,10).
1.3 Cattle spermatogenesis
Spermatogenesis is the cellular transformation within the seminiferous epithelium of the adult testis which results in spermatozoa production, a finely regulated process of germ cell multiplication and differentiation. The process is made up of three main processes: Mitotic replication of spermatogonia, meiosis, and postmeiotic differentiation into spermatozoa (11).
1.4 Fertility of frozen and fresh semen
Fresh semen will always provide the better semen evaluation, but with the increase in artificial insemination there is also an increase in frozen semen. The first calf produced from frozen-thawed semen was in 1951, showing that frozen semen was and is still fertile when properly preserved (12). The fertility of the spermatozoa is of outmost importance, since it is its primary focus and function of the bull and subsequent insemination. It is therefore important to know the difference of the fertility when it is fresh, and when it has been cryopreserved for an extended amount of time. Cryopreservation results in poorer fertility when inseminated compared to equivalent amount of fresh semen (13). It reduces the fertility and has also shown to contain more perinuclear theca pathologies among others. Improving the knowledge about the pathogenesis, prevention and improvement of the efficiency of semen preservation can be made, such as that the change of temperature changes the sperm plasma membrane composition and structure, and with that its function (14). Over the years, the fertility of frozen semen has been improved by the development of better diluents, freezing
10 protocols, viability testing, extenders, and a generally better understanding of the cryopreservation related spermatozoa damage (8).
1.5 Thawing effect on quality
Frozen semen can experience damage during thawing process if it is a too slow or too long thawing process. During too slow thawing, ice crystals can form and be harmful to the spermatozoa. Caution should also be to not overheat the sample as harm can be seen then as well. A temperature of 37°C for 10-45 seconds is considered optimal as it limits the time for recrystallization in the spermatozoa. Crucial factor is storage and handling of semen straws - usually packaged in 0.25-0.5ml straws. The need for proper care in storage and handling of these tanks cannot be overemphasized. If not, compromised semen quality and reduced pregnancy rate can be seen (8).
1.6 Breed standards
There is a difference in fertility and quality of semen depending on the breed used, where a study showed Aberdeen Angus and Simmental as 2 breeds out of 17 that had a much higher chance of being above the requirements of being satisfactory compared to Hereford, Limousin, Charolais breeds, according to BSE standards (15).
1.7 Heritable measurements
The two types of bulls are separated for their intended use - dairy cows, and beef cows. They both have a common goal of having a high fertility rate, and to improve the next generations output, and while some of the factors affecting that are environmental and managemental, some are also heritable. Genetic selection to improve the next generation is possible, and some genetic correlations can be seen with output traits such as milk production or growth rate. The information about the genetic is included in many breeding programs of both dairy and beef, and has shown a clear advantage in genetic gain (16).
11
1.8 Method of semen collection
The most common methods of semen collection from bulls are by artificial vagina or electro-ejaculation. When collecting via the artificial vagina, the semen is collected when the bull has not received food recently due to lowering of the sex drive when full. The collection is done in a room called “manage” that fulfills the appropriate sanitary and health standards. The bull will be mounting a dummy cow or a teaser, where a handler will direct the penis by the prepuce to the inlet of the artificial vagina. Directly after collection the semen should be evaluated (17).Electroejaculation is the process of inserting a probe into the rectum of the bull and applicating a short, low-voltage pulse of electrical current to the nerves of the penis to stimulate the ampullae as well as vas deferens to induce ejaculation. It is a convenient, quick, and reliable method for collection of semen but has the potential to cause discomfort and should not be preferred over less invasive procedures when possible. If it is necessary, the procedure should be done in a manner as to minimize discomfort for the bull, and to provide pain relief, sedatives, or epidural anesthesia when possible (18).
1.9 Andrological Examination
1.9.1 Morphological examination of external genitaliaThere are multiple variables when examining the bull before breeding - one of which is the external genitalia. The scrotum should be palpated for any abnormalities in the testes or epididymides that can be found and that might affect the fertility rate or health of offspring of semen. The bull should be palpated rectally to evaluate the accessory sex glands, which is done systematically and with care to avoid damage and lesions, but also to identify certain pathologies such as epididymitis, inguinal hernias, or vesiculitis. The sheath of the penis is palpated for any preputial infections, damage, or injuries to the penis which can be seen when the tip is visualized. The testicles do not need to be completely identical from bull to bull, as each might have some slight anatomical differences and variations in size, position, as well as shape of testicles. It should however be noted, that some breed society pre-sale inspections bulls are not allowed for any variations in size and shape. A major part of the examination in the external genitalia of bulls is the scrotal circumference, as the size is a good indicator of sperm output. The size is highly correlated with the daily output of sperm and is a constant figure, where the minimum standard for certification for certain breeds are 32-34cm. Being underneath the standard for the breed evaluated can be considered having testicular hypoplasia and being sub fertile (19-21).
12 1.9.2 Macroscopic semen evaluation
When evaluating semen macroscopically, the color and consistency is considered. The sample should be white as well as opaque. Some samples might have a hint of yellow or red, in case of urine contamination or hemorrhage respectively. The consistency of the sample is dependent on the concentration of the semen. The more concentrated it is, the creamier the sample will appear, and it will appear more liquid if it is less concentrated (21,22).
1.9.3 Microscopic semen evaluation
1.9.3.1 Subjective motility determination
The gross motility is evaluated by placing a non-diluted sperm drop sample on a pre-heated slide under 100x magnification. The progressive motility can be evaluated by diluting the semen sample and examining it on warmed slides as well as coverslips under a 100-400x magnification. Progressive motility score should generally be above 60 percent, otherwise it is deemed unsatisfactory. However, depending on which standard or certificate that is sought after the gross motility needs to be between 30-60 percent individual progressive motility (23). Penny’s (2010) authors believes that if the sample is evaluated on a heated pad as well as handled properly, most bulls will have an individual progressive motility of above 60 percent (21).
1.9.3.2 Computer-assisted sperm analysis
CASA (computer-assisted semen analysis) is a computerized imaging system used to visualize the sperm and that uses a certain type of software program to evaluate dozens of types of individual sperm parameters. The semen sample is placed on the slide holder of the microscope, which has a high-resolution video camera attached to it. The camera then feeds the magnified data to the computer where it can be analyzed by the software. There are currently many different types of CASA systems marketed by different companies (24). The sperm in the sample is visualized by a dark field/negative phase contrast/fluorescent optics microscope. Parameters for the CASA system for analysis depends as much on the operator’s expertise and training as the settings of the system such as calibrations, validations, standardization, and optimization (25).
13 These systems, when used correctly, can provide a precise and accurate picture of the sperm motion characteristics, however, morphology has as of the year 2002 been poorly studied and more are warranted (26).
1.9.3.3 Spermatozoa concentration and total sperm count
A both important as well as fundamental step in evaluation of spermatozoa for artificial insemination. Can be done poorly and without accuracy by visual inspection, or evaluated with for example Sysmex particle counter, Karras Spermiodensimeter and with Neubauer haemocytometer which is considered the gold standard for evaluation of the sperm concentration (27). It has also shown that AI organizations reported that the average cryopreserved AI dose contains approximately 20 x 106 total spermatozoa (with a range of 10-40 x 106), a 2-20 times greater difference than that of the minimal threshold for normal fertilization (28).
1.9.3.4 Morphology
Morphologically healthy sperm should be above or equal to 70 percent, and is considered the norm internationally, otherwise marked as unsatisfactory. It should be carried out in all bulls by counting 100 cells under a 1000x oil immersion magnification and recording their percentage of defects detected. The slides should be prepared with a nigrosine-eosin stain. If there were to be a close margin to the standard threshold, a second count should be done (29). A study showed that the percentage of proximal droplets (p<0.01) as well as "primary" abnormalities (p<0.01) had a significant effect on the bull’s fertility. (30) Abnormalities that are found vary in prevalence depending on the bull, but also on the time of evaluation, and vary between head defects, midpiece defects, coiled principal pieces, as well as proximal droplets and other types of defects found. The semen should be handled under optimal laboratory conditions, acceptable progressive motility can be seen in cases with good sperm morphology (15).
1.9.3.5 Viability
Viability examination can be done either by using a nigrosine-eosin stained sperm smear to identify each live or dead spermatozoa, or by doing an individual progressive motility assessment to estimate the percentage of viable sperm. Where the later one is a much faster and convenient way,
14 and the former a more methodic and thorough method (21). There is also a high correlation between motility and viability seen (15).
1.10 Insemination techniques
There are two methods of insemination in cows: artificial insemination and natural mating. Artificial insemination holds the benefit of being cost-effective, improve the genetics of the herds output and reproductive performance, being able to use worldwide semen of best quality, adjust it to the cow’s estrus, minimize risk of damage and transmissible diseases to the cow and bull, and has been available and improved upon for over 60 years. The market for global artificial insemination of various animals was estimated to USD 3.79 billion and is expected to reach USD 3.95 billion in 2020 where cattle segment accounted for 46.5% in 2019, where UN FAO in 2018 published data that beef and buffalo production increased by 2.05 million tons from 2017 to 2018 alone. The market price and popularity can be attributed to the growing artificial insemination market worldwide, where Asia Pacific accounted for 30% in 2019 due to its large cattle population, as well as an increased consumption of animal protein, growing demand for cattle with high daily gain or milk yield. The reduced cost of housing, handling the bull, genetic selection, and increased safety for the farmer and animals can also be regarded as advantages of artificial insemination over natural breeding (31).
1.11 Semen Storage
If the semen of the bull is not to be used immediately – fresh - it can be saved and stored for later use or selling by preserving it frozen in liquid nitrogen – cryopreservation. The storage process for cryopreservation consists of three to four main procedures: dilution, cooling, and freezing/thawing. The semen is stored in straws of two main sizes, 0.25ml and 0.5ml, with the identity information labeled on the side. Fresh semen can also be stored in both of these straws, but normally only the 0.25ml is used then. The sperm is diluted and mixed with sperm protectants, such as buffered egg yolk or heated milk. Glycerol is added as a special cryoprotectant and storage temperature for the samples is -196°C in liquid nitrogen (32).
15 1.11.1 Frozen semen
It has been shown that frozen semen compared to the fresh semen has its fertility reduced, and that freezing decreased the viability and fertility by up to 50-70% (33). A common cryoprotectant
over the years has been glycerol, allowing the sperm to be saved for months, years, or decades. As the semen can be preserved for years by cryopreservation, quality checks can be performed quarterly or every six months, as well as an initial evaluation before freezing of its viability and motility for refence, filtering, and comparison. Random sampling from each batch is taken and examined for bacterial load, hypo osmotic swelling test, thermo resistance test, percent intact acrosome, and concentration (34).
1.11.2 Semen extenders
Semen extenders can be considered the main constituents to accomplish freezing and preservation with minimal damage. They provide the semen with energy, maintain the osmolarity, reduce contamination from bacteria as well as protect it at low temperatures. Liquid semen extenders are diluents that are added to the semen sample to preserve it. It is a buffer and protection to the sperm cells from their own byproducts, shielding it from cold and osmotic shock as well as preserving it. Different extenders can be used. The extenders buffers the semen to maintain pH of the medium (such as tris, sodium phosphate, and citric acid), and has added cryoshock preservatives (such as glycerin, egg yolk, milk, or soy-lecithin) to provide energy for the sample (fructose) and antibiotics can be added to reduce microbial contamination (streptomycin, penicillin) (35).
16
RESEARCH METHODS AND MATERIALS
The research work was carried out in the Animal reproduction laboratory of Large animal clinic of Lithuanian University of Health Sciences (LUHS) in Kaunas, Lithuania. The samples were provided frozen by the laboratory and the examination procedures were undertaken during May-June of 2020. In the study, semen samples of 23 bulls were evaluated, where 9 out of the 23 samples were from beef bulls and 14 from dairy bulls, both of different breeds. The mean age of bulls were 61.37 ± 3.92 months.
The information about the samples were obtained from the companies that sent the samples to the lab, such as their name, age, breed, type of bull, and fertility rate.
The semen was collected frozen from liquid nitrogen storage cylinders which are stored at the Animal Reproduction Laboratory, which contained about six-seven doses/straws from each bull. Three doses of the same ejaculate and batch were retrieved for analysis. The doses were all thrown into a water bath of 37 °C. Two out of the three doses were taken out after a timed ten seconds, the tips of the straws were cut and semen samples were placed on microscopic slides to evaluate directly after thawing (0 hours): the motility (both subjectively by eyes as well as objectively by SCA-program), viability (with eosin/nigrosine staining method), concentration, as well as morphology. The third out of the three doses were taken out of the water bath after a timed five hours of water bath incubation had passed (5 hours). The tip of that straw was cut, emptied on a microscopic slide, and assessed and analyzed for its motility (subjectively and objectively) as well as the viability. Since the morphology and concentration does not change between the straws due to being from the same batch and ejaculate, it was only measured once. 23 samples of concentration and morphology was examined, as well as 46 samples of motility and viability.
2.1 Motility assessment
The motility of the dairy and beef sperm was performed subjectively – visually via microscope – as well as objectively – via SCA (Sperm Class Analyzer (SCA, Microptic, Spain)) computer assisted sperm analysis program. The motility was determined at 37 ± 0.5 °C due to maximum activity of the sperm with a phase contrast microscope (Nicon ECLIPSE 50i, Japan) that contains a built-in heating table to ensure no temperature drop or fluctuation during analysis, along with heated lenses, coating, and pipette tips to again assure the optimal temperature for the sperm. The sperm was collected from a straw that was placed in a water bath (Memmert, Germany) for 10 seconds to reach the temperature of 37.0 ± 0.5 °C after retrieving it from the liquid nitrogen storage container. After thawing, a drop
17 of warm sperm (5–10 µl) is collected and placed on a 37 ± 0.5 °C heated slide and covered with equally warm liquid.
The subjective motility of the sperm samples was measured under the microscope with the naked eye, determining within a three to five field view the percentage of progressive spermatozoa observed. The final result for each sample is calculated according to the arithmetic mean of all measurements taken.
The objective motility uses the same microscope as for subjective motility, and is determined with the help of a chamber. The image from the microscope is converted and transferred to a digital image on a computer linked to the microscope, installed with a sperm motility evaluation system SCA (MICROPTIC S.L., Spain). Each of the 23 samples was evaluated twice – at zero and five hours post thawing - in at least three fields of vision, which gives the computer program enough values to calculate and record the total percent sperm motility (Motility SCA) and the percent of progressive motility (Progressive motility).
2.2 Viability assessment
Viability of each sample was examined by dying them with Eosin-Nigrosine dye. One drop (5 – 10 µl) of diluted semen is placed on a pre-heated (37°C) slide and mixed with 20, 10 µl of eosin, nigrosine dye respectively, letting the smear air-dry for a few minutes. The smear should be mixed carefully and thinly as to not create overlapping and create a better microscopic view. The sample is examined with a phase contrast microscope (Olympus BH2, Olympus Optical CO., Ltd., Japan) under 1000x magnification with immersion oil. A total of 200 sperms – for accuracy - were counted and recorded by differentiating between alive (white) or dead (pink or red) sperms in the dark background of nigrosine dye. The live sperms appear white in the smear due to that the eosin only can penetrate cracked, ruptured, or otherwise damaged sperm membranes creating a reddish appearance, while the membrane of healthy sperm remain intact and no eosin dye can enter. After the allotted number of sperms are counted the viability is calculated by the percentage of alive spermatozoa compared to the total number.
2.3 Sperm concentration
With the help of a phase contrast microscope (Nicon ECLIPSE 50i, Japan), a 100-fold magnifying lens, and a haematocytometric (Neubauer) camera, the concentration of each sperm
18 sample is measured. The semen samples are diluted to a ratio of 1:20 with distilled water and thoroughly mixed and inserted into the haematocytometric chamber. The sperm is calculated by counting each sperm cell in five large squares (80 small squares), or 0.2mm2, three times in three different squares in the same chamber. The mean value is taken from the result of those calculations. The total concentration of the sperm sample is then calculated according to the formula listed below:
K = S × P × 5 × 10 × 1000, where:
K – Concentration of the sperm sample (number of spermatozoa per milliliter) S – Total number of sperm cells in 80 small squares
P – The dilution ratio
5: The coefficient as sperm were counted in a 0.2mm chamber area 10: From the depth (0.1mm) of the chamber and is multiplied 1000: Used to multiply to convert to 1 ml
2.4 Morphological evaluation
The determination of sperm head and tail pathologies, both which are counted and recorded separately in the same sample.
Each sample is stained with methylene blue dyes according to the instructions given by the manufacturer (Sperm Blue, MICROPTIC S.L., Spain) to evaluate the pathologies.
Sperm Blue Sperm dying:
1. The lens is smeared as well as air-dried
2. The slide is placed in the fixative solution (10min) 3. Then it is placed in the dye (20-30min)
4. Immerse the painted glass in distilled water (3 seconds) and air dry
The dyed slide is observed with a phase contrast microscope (Nicon ECLIPSE 50i, Japan), using the 1000x magnification with immersion oil. Count 500 spermatozoa from different parts of the slide – for variability and accuracy – and identify its morphological head pathologies. The head pathologies that are identified and recorded are: pear-shaped, narrow-headed, abnormal contours, underdeveloped, abnormal heads without tails, narrow heads, large heads, small normal heads, short broad heads, and spermatozoa with paracentric attachment to the middle-part. After the spermatozoa has been counted, each pathology can be calculated for its total percentage of pathological heads.
To evaluate the tail pathologies, the wet preparation (Hancock method) was used. The sperm sample was diluted with 1:10 buffer formalin solution. After the dilution one drop (5-10 μl) is placed
19 on a slide with a cover slide. Allow the sample to settle for several minutes before evaluating it, which is done with a phase contrast microscope (Olympus BH2, Olympus Optical Co., Ltd., Japan), using the 400x magnification. Count 200 spermatozoa from different parts of the slide. The following sperm tail pathologies are identified and recorded: mid-abnormalities, usually and under the head and double tails, proximal drops, distal drops, sperm heads without a tail, acrosome defect, acrosome lesions, vacuoles. After the counting, each pathology is calculated for its total percentage of all pathological tails. The final evaluation is to calculate the total percentage of all pathologies, i.e. heads, tails, other from all evaluations.
2.5 Statistical analysis
Statistical analysis was calculated and performed with the programs Microsoft Excel and IBM SPSS Statistics 20 for Windows 10 (SPSS for Windows 10.0, SPSS Inc., Chicago, IL, USA). The data included and presented were analyzed via Independent-Samples T Test and Paired-samples T test, comparing the means ± SD, Std. error mean, and their differences to each type of bull. The slope of line for each graph was calculated by dividing the difference of the y-axis values of two points to the difference of the x-axis values of the same two points. The differences in each calculation was considered to be of significance when: *p<0.05; **p<0.01; and ***p<0.001.
20
RESEARCH RESULTS
3.1 Sperm quality values and descriptive results at 0 hours and after 5 hours
3.1.1 Motility between two cattle typesThe sperms mean motility was measured at two different times: at 0 hours, and 5 hours after thawing, between two different type of bulls: beef and dairy. Each sample was measured for subjectively, by SCA, and for progressive motility. There was generally no significance calculated between the two types (p>0.05). The mean subjective motility for beef at 0 hours measured at 53.89 ± 4.55%, while for dairy at 0 hours it measured 56.79 ± 4.88% (Fig. 1). A mean difference of 2.9 ±
6.67% (p>0.05) was measured. The means measured 33.33 ± 5.20% for beef at 5 hours after thawing, while 34.57 ± 4.08% for dairy. With a mean difference of 1.238% (p>0.05). The results were not considered significant. The beef value at 0 hours measured 20.56 ± 3.86% higher than at 5 hours (p<0.001) and the slope of line for beef was -4.112. At 5 hours after thawing, the dairy mean value had decreased by 22.21 ± 2.98% (p<0.001) with a slope of line of -4.443. Both differences were significant.
When SCA was used it measured the highest mean motility for beef at 0 hours 84.63 ± 3.61%, while for dairy at 0 hours 85.44 ± 3.80%. A mean difference of 0.88 ± 5.24% was measured (p>0.05).
0 10 20 30 40 50 60 P erc ent %
Beef 0 Beef 5 Dairy 0 Dairy 5
Fig. 1 Mean motility value measured for subjective motility showing beef and dairy bulls at 0 hours and at 5 hours after thawing
21 The means measured 66.38 ± 4.54% for beef at 5 hours after thawing, and 62.05 ± 5.39% for dairy, with a mean difference of 4.33 ± 7.05% (p>0.05) (Fig. 2). At 0 hours, the beef value measured 18.25 ± 4.26% higher compared to at 5 hours (p>0.001) and the slope of line for beef was -3.65. At 5 hours
0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 P erc ent, %
Beef 0 Beef 5 Dairy 0 Dairy 5
Fig. 2 Mean motility value measured by SCA showing beef and dairy bulls at 0 hours and at 5 hours after thawing
0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 P erc ent, %
Beef 0 Beef 5 Dairy 0 Dairy 5
Fig.3 Mean motility value measured for its progressive motility showing beef and dairy bulls at 0 hours and at 5 hours after thawing
22 after thawing, the dairy mean value had decreased by 23.38 ± 3.27 % (p<0.001) with a slope of line of -4.678. The difference in SCA motility for dairy was considered significant.
When the sample is measured for its progressive mean motility for beef at 0 hours, it measured 57.73 ± 5.26%, while for dairy at 0 hours it measured 60.85 ± 5.69%. A mean difference of 3.12 ± 7.75 % (p>0.05). The means measured 44.93 ± 4.67% for beef at 5 hours after thawing, and 45.39 ± 4.9%, with a mean difference of 0.46 ± 6.77% (p>0.05). The mean difference decreased by 12.8 ± 5.05% for beef (p>0.01) from 0 hours to 5 hours, and 15.46 ± 5.44 for dairy. (p>0.01). The slope of line was measured at -2.559 for beef and -3.092 for dairy. (Fig. 3).
When non-progressive mean motility is measured for beef at 0 hours, it measured 26.79 ± 2.56%, while for dairy at 0 hours it measured 24.58 ± 2.75%. A mean difference of 2.21 ± 3.75% was measured (p>0.05). The means measured 21.45 ± 2.66% for beef at 5 hours after thawing, while dairy measured 20.95 ± 2.01%, with a mean difference of 0.51 ± 3.33% (p>0.05). The mean difference decreased in beef by 5.34 ± 3.21% (p>0.01) and in dairy 3.64 ± 3.75% (p>0.01). The slope of line in beef bulls were -1.067 and in dairy -0.727. (Fig. 4).
When measured for its immotility the mean for beef at 0 hours measured at 15.49 ± 3.66%, while for dairy at 0 hours it measured 15.78 ± 4.32% and with a mean difference of 0.29 ± 5.66% (p>0.05). The means measured 33.62 ± 4.54% for beef at 5 hours after thawing, while dairy measured 37.95 ± 5.39% at 5 hours, with a mean difference of 4.33 ± 7.05% (p>0.05). The mean difference
0,00 5,00 10,00 15,00 20,00 25,00 30,00 Per ce nt, %
Beef 0 Beef 5 Dairy 0 Dairy 5
Fig. 4 Mean motility value measured for its non-progressive motility showing beef and dairy bulls at 0 hours and at 5 hours after thawing
23 increased in beef by 18.13 ± 4.22 (p>0.001) and 22.17 ± 2.63% in dairy (p<0.001), and was considered significant. The slope of line in beef was measured at +3.626 and in dairy +4.434 (Fig. 5). 3.1.1 Viability between two different cattle types
The viability mean was measured at 51.00 ± 2.24% for beef bulls at 0 hours and 58.5 ± 2.26% for dairy bulls at 0 hours with a mean difference value of 7.5 ± 3.18 (p>0.01). At 5 hours after thawing,
0,00 5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 Per ce nt, %
Beef 0 Beef 5 Dairy 0 Dairy 5
Fig. 5 Mean motility value measured for immotility showing beef and dairy bulls at 0 hours and at 5 hours after thawing
0 10 20 30 40 50 60 70 0 5 P erc ent, % Hours Beef Dairy
Fig. 6 Mean sperm viability value measured showing beef and dairy bulls at 0 hours and at 5 hours after thawing
24 beef sperm measured 33.78 ± 3.09% and for dairy 34.36 ± 1.97%. The mean difference between them were measured to 0.58 ± 3.66% (p>0.01). The mean difference decreased in beef by 17.22 ± 1.98% (p<0.001) and in dairy 24.14 ± 2.87% (p<0.001) and are both considered significant. The slope of line for beef bulls were -3.444 and for dairy -4.828 (Fig.6).
3.1.2 Concentration between two different cattle types
The mean concentration/dose for beef type was measured at 19.46 ± 2.12%, and for dairy it measured 21.01 ± 1.29%. The mean difference was measured to 1.55 ± 2.33% (p>0.05). (Fig. 7).
The mean concentration/ml for beef was measured at 77855556 ± 8463567 mil/ml and for dairy 84035714 ± 5179835 mil/ml. The mean difference was measured at 6180158 ± 9334343 mil/ml (p>0.05). The results were not considered significant. (Fig. 8).
19 19 20 20 21 21 22 Beef Dairy Mill ions
Fig. 7 Mean concentration/dose value measured showing beef and dairy bulls
74000000 75000000 76000000 77000000 78000000 79000000 80000000 81000000 82000000 83000000 84000000 85000000 Beef Dairy C on ce nt ra ti on /m l
25 3.1.3 Morphology of different cattle types
The morphological pathologies in beef and dairy bull’s sperm heads are divided into 10 groups. Most frequently found in beef, as well as in dairy, were abnormal contour pathologies (Fig. 9-10).
Compared to the second most found pathology in the head - pear shaped in beef and narrow base in dairy - the most frequently found is 30 and 39% more commonly found in each bull type respectively (p>0.05). There were more head pathologies found in beef than dairy bulls. In beef there
18% 5% 48% 4% 5% 10% 5% 5% 0% 0% Pearshaped Narrow base Abnormal contour Undeveloped
Loose abdormal head Narrow head
Big head Small Short wide Paracentrical
Fig. 9 Morphological sperm head pathologies found in beef bulls
5% 13% 52% 9% 6% 9% 6% 0% 0% 0% Pearshaped Narrow base Abnormal contour Undeveloped
Loose abdormal head Narrow head
Big head Small Short wide Paracentrical
26 were 8.11 ± 2.14% (p>0.05) pathologies found while in dairy 6.64 ± 1.32% (p>0.05) pathologies. A mean difference of 1.47 ± 2.38% was measured (p>0.05) (Fig. 11-12).
The morphological pathologies in beef and dairy bull’s sperm tail and others are also divided into 10 groups. In beef, the most frequently found pathology was simple bent with a measured value of 33%, while in dairy it was loose head with 31%. (Fig. 13-14).
0 1 2 3 4 5 6 7 8 9 Beef Dairy P erc ent, %
Fig. 11 Mean pathology values of sperm head pathologies found in beef and dairy bulls
0 10 20 30 40 50 60 70 80 90 100 Pathologies Normal P erc ent, % Beef Dairy
27 Compared to the most frequently found pathology, the second most frequent was 5% less common in beef (loose head) and 8% less common in dairy (simple bent) (p>0.05). There were more tail and other pathologies found in beef than dairy. In beef there were 9.78 ± 1.78% (p>0.05) pathologies found while in dairy 7.86 ± 0.98% (p>0.05). A mean difference of 1.92 ± 2.04% was measured (p>0.05) (Fig. 15). 10% 11% 28% 0% 0% 0% 2% 33% 11% 5% Proximal droplet Distal droplet Loose head Acrosome defect Abnormal acrosome Vacuole Abnormal midpiece Simple bent Tail coiled Double coiled
Fig. 13 Morphological sperm tail and other pathologies found in beef bulls
7% 11% 31% 0% 0% 0% 9% 23% 11% 8% Proximal droplet Distal droplet Loose head Acrosome defect Abnormal acrosome Vacuole Abnormal midpiece Simple bent Tail coiled Double coiled
28 There were 1.67% more pathologies measured in the sperm tail and other, compared to the sperm head in beef (p>0.05). There were 1.22% more pathologies in the sperm tail and other compared to the sperm head in dairy (p>0.05) (Fig.15-16).
0 2 4 6 8 10 12 Beef Dairy P erc ent, %
Fig. 15 Mean values of pathological and normal sperm tail and other found in beef and dairy bulls 0 10 20 30 40 50 60 70 80 90 100 Pathologies Normal P erc ent, % Beef Dairy
Fig. 16 Mean values of pathological and normal sperm tail and other found in beef and dairy bulls
29 3.1.4 Fertility rate between two different cattle types
The mean fertility rate for beef bulls were measured at 44.18 ± 3.86% and 48.6 ± 1.94% for dairy bulls. The mean difference of 4.42 ± 3.91% was measured and considered significant (p<0.05) (Fig. 17). 41 42 43 44 45 46 47 48 49 Beef Dairy P erc ent, %
30
DISCUSSION OF RESULTS
Choosing the correct sperm sample for artificial insemination to improve the quality of your herd or farm is a vital step in farming. Two different types of bovine were evaluated for their sperm quality: dairy and beef. A study by Berry and co-authors (16)showed that semen quality in bulls has become a point to improve, since it has shown that even if the cow side of the fertilization could be improved, fertilization rates have also improved when using a high-quality sperm sample from the bull.Dairy bovine shows a higher overall quality in sperm and fertility compared to beef bulls, which can be seen in the study of Morrel and its other authors (36). The study also showed that there was increased viability in dairy bulls and that there were no significant differences in total and progressive motility between the two bull types. The same study showed that dairy bull semen measured overall better in fertility values compared to beef bulls, which also corresponds to the findings of this study (36).
For measuring fertility and its quality for breeding, it is important to evaluate morphology and motility as they are important parameter for semen quality assessment (30,37) where said qualities (motility, morphology) can relate to over 70% of problems identified in not-suitable for mating bulls.
The motility should be evaluated with a minimum threshold of 60%, where only those above the threshold are considered satisfactory (30).
A study by Madison and others showed that improving the semen quality measures (motility and morphology in this study) could increase production, improve efficiency, and provide benefits and profitability (38,39).Thun and others (40) showed that cryopreserved dairy bull semen that were observed and measured in three different type of extenders, where 25, 32, 43% out of samples from those three extenders did not go above 50% motility subjectively, progressively and were therefore measured as unsatisfactory. The measured mean percentage for motile sperm directly after thawing in each sample was 74.7 ± 6.9, 73.4 ± 7.6, 69.1 ± 6.9%. The normal, healthy sperm morphology percentage for each sample was measured at 67.5 ± 8.0%, 65.4 ± 9.4%, 59.0 ± 10.5%. The fertility rate for each sample was between 57.65 ± 8.05 and 69.07 ± 5.91%, depending on which breed and extender that was used. Gil and other authors (41) thawed their samples of dairy semen in a water bath (35º C for 12s), measured the subjective motility post-thaw where the samples needed 70% or above to fulfill the minimum standards. CASA was also used to measure the motility and was done immediately after thawing and after six hours of incubation in 20-22º C. Fertility rate in two mediums: 69.1 ± 0.8, 69.2 ± 0.8 post-thaw. The subjective motility was measured at 56.0 ± 0.8 directly after thawing, and was significantly higher than those after six hours. Hinsch and other researchers (42) compared semen parameters in two different extenders in dairy bulls, the samples complied with
31 official standards of motility and morphology when evaluated, and had a post thawing incubation time of three hours at 4º C. The thawing was performed at 38º C for 25s. Analysis of motility was done with a CASA program, where the total motility of freshly diluted semen was 91.4 ± 3.3%, directly after thawing 61 and 60%, and after eight hours of incubation it measured 41 and 40%. The shown fertility rate was 68.3% and 67.8% in each extender sample.Van Wagtendonk-de Leeuw and others (43)studied the difference in three different extenders in dairy bulls, with measured subjective motility means of 41.9 ± 0.5%, 42.2 ± 0.5%, and 39.2 ± 0.5%. Kaka and other scientists’ (33) study of beef bulls evaluated via CASA in 37º C water bath for 30s after cryopreservation, measured a mean value of 40.00 ± 4.0 %. The viability was measured at 59.75 ± 1.0%. The mean of normal morphology was measured at 59.5 ± 4.3%. The mean viability was measured at 53.1% in frozen-thawed samples in another study which also showed that freezing-thawing reduced the percentage of motile and alive spermatozoa (14).
This study measured values of subjective motility which for beef measured 53.89 ± 4.55% and for dairy 56.79 ± 4.88% directly after thawing, and after five hours measured 33.33 ± 5.20% and 34.57 ± 4.08% for dairy. The results were above the values of Van Wagtendonk-le Leeuw and others (43) as well as Gil (41) studies reported, but below those of Thun and others (40). By SCA, it measured in beef 84.63 ± 3.61% and 85.44 ± 3.80% for dairy directly after thawing. The means measured 66.38 ± 4.54% for beef after five hours, and 62.05 ± 5.39% for dairy. These results are in line with what the other studies showed and above the established thresholds. Viability was measured at 51,00 ± 2.24% for beef bulls directly after thawing and 58.5 ± 2.26% for dairy bulls. After five hours, beef sperm measured 33.78 ± 3.09% and for dairy 34.36 ± 1.97% which for dairy is in line with the study of Kaka and co-authors (33), while beef is a bit lower. The results is also similar to that of Martinez and others (14). The mean concentration/ml for beef was measured at 77,855,556 ± 8,463,567 mil/ml and for dairy 84,035,714 ± 5,179,835 mil/ml, which is an acceptable mean as well as above the value threshold for cryopreserved semen (28). The mean fertility rate for beef bulls were measured at 44.18 ± 3.86% and 48.60 ± 1.94% for dairy bulls, which is a bit lower compared to the above-mentioned studies. The normal spermatozoa according to morphology was always above 90%, which was better than reported studies.
The study was overall in line with the mentioned studies, better in some ways while worse in others, such as a reported fertility rate below that of the other studies. Meanwhile, the other parameters are following the other studies. A note from author is that this study did not take into consideration the cryopreservation nor which type of breed was used in its calculations compared to some of the mentioned studies.
32
CONCLUSIONS
1. The motility had overall best measurements in dairy bull semen - both directly after thawing and after five hours of incubation. The difference measured for its subjective, SCA, and progressive motility directly after thawing was 2.9 ± 6.67%, 0.88 ± 5.24%, and 3.12 ± 7.75% (p>0.05). After five hours, the subjective and progressive motility were also higher (1.24 ± 6.58% and 0.46 ± 6.77%) in dairy bull semen (p>0.05). After five hours, the SCA motility was measured 4.33 ± 7.05% higher in beef bull semen (p>0.05).
2. The viability of spermatozoa was higher in dairy bull semen than in beef semen, both directly after thawing, (58.5 ± 2.26%) (p<0.05) as well as after five hours (34.36±1.97%) (p>0.05).
3. The highest concentration per dose was seen in dairy bull sperm (21.01 ± 1.29 mil/dose) with a mean difference of 1.55 ± 2.33 million (p>0.05).
4. Evaluation of beef and dairy bull semen morphology showed that both bull types semen was of good quality and did not exceed the maximum allowed pathologies in the dose. The percentage of sperm head pathologies detected in beef bull semen was 1.47 ± 2.38% lower than in dairy semen (p>0.05), and both beef and dairy had abnormal sperm head contour as the most common pathology. The percentage of tail and other pathologies in dairy bull semen was higher by 1.92 ± 2.04% (p>0.05).
5. Dairy bull sperm was evaluated with the highest fertility rate of 48.6 ± 1.94%, with a mean difference between the two types of 4.42 ± 4.32% (p<0.05).
33
RECOMMENDATION
Dairy bull semen showed higher measurements and can therefore be said to be better than beef bull semen only from the measurements evaluated. However, dairy and beef bulls are used in their specific areas and their sperm is then normally not comparable. The person purchasing or acquiring bull semen should instead always pursue the highest quality of sperm as well as fertility rate, as the studies mentioned showed that if the quality of the sperm is high the higher will the fertility rate and subsequently the success rate of each insemination be.
34
ACKNOWLEDGEMENT
I would like to express my most sincere and deepest appreciation and gratitude for my supervisor Dr. Neringa Sutkevičienė, as her continued support, kindness, knowledge, and reliability has been a corner stone of my master thesis. She took her role as a supervisor above and beyond and I could not have asked for a better outcome and I will be forever thankful for her dedication towards helping me.
Furthermore, I would like to acknowledge the Veterinary Academy of Lithuanian University of Health Sciences for providing me with the necessary education, information, and supplements towards finishing this master thesis.
Lastly, I would like to express my gratitude to my friends and family for their continued love and support, for which I am greatly thankful for.
35
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