Recent trends in semen collection, evaluation and cryopresevation

AK Misra and S Tyagi

Project Directorate on Cattle
Grass Farm Road, Meerut Cantt, Uttar Pradesh 250 001

Introduction
Much work has been done on semen preservation within the last fifty years, however, no full
proof freezing process has been standardized so far. The spermatozoa behavior during freezing
process from different species and breeds within the species still differ in many respects. The
problem of significant differences among bulls with respect to the sensitivity of their spermatozoa to
cryopreservation still remained to be solved.
The first reliable techniques used to obtain motile bovine spermatozoa after freezing and
thawing were described by Smith and Polge in 1950 (Smith and Polge, 1950a;1950b). In the next
year, the birth of the first calf, born as a result of artificial insemination with spematozoa
cyopreserved by the method of Smith and Polge, was reported (Stewart, 1951). Even after 50 years
of extensive research, the greatest problem in sperm cryopreservation is the poor recovery of
progressively motile spermatozoa after freezing and thawing. Bull ejaculates carefully selected as
per the standard quality parameters before freezing may yield only 30-40% motile sperm after
thawing (Foote et al., 1993) and 5-15% of ejaculates from bulls meeting the standards for processing
are discarded after freezing due to substantial loss of sperm motility (Bearden and Fuquay, 1997).
The great variability among bulls with respect to the sensitivity of their spermatozoa to the rigors of
processing and freezing particularly for cross bred bulls resulted in culling of a large number of
breeding bulls regardless of their genetic potential.

The artificial insemination had tremendous impact on the dairy industry. Extensive
distribution of semen from genetically valuable bulls, resulting in rapid improvement in the
international dairy industry could become possible due to storage of semen in the frozen state. The
increase in the productivity of dairy cattle over a relatively short period of time has been attributed to
rigorous genetic selection of bulls and use of cryopreserved spermatazoa from these bulls for Al
(Foote, 1982). Once a bull is identified as producing daughters with superior milk production,
thousands of doses of his semen can be cryopreserved and used to inseminate large numbers of cow,
thus producing large number of productive daughters.
Progress in semen collection and dilution, and cryopreservation techniques now enables a
single bull to be used simultaneously in several countries for up to 100,000 inseminations a year
(Gibson and Smith 1989). This implies that a very small number of top bulls can be used to serve a
large cattle population.

Semen Collection
The main objective of any Artificial Insemination Organization is to produce the largest quantity of
the highest quality of semen possible in an efficient amount of time (Pennington, 1990). Since semen
ejaculation is a physiological process governed by a variety of stimuli besides the neuro humoral
factors, the quality of resultant ejaculate is greatly dependent on the sequence of steps used
commencing from sexual preparation of the bull to final ejaculation. So the effective and reliable
methods of semen collection would allow the germplasm stations to produce more and more number
of frozen semen doses for developmental activities.

Sexual Preparation
Sexual preparation means prolonging stimulation of the bull beyond that needed to induce mounting
and ejaculation. Proper sexual stimulation followed by restraint enhances the contractions of the
muscles involved in emission and ejaculation of semen. Several studies have confirmed that the
sexual performance of bulls was enhanced by allowing them to view their counterparts engaged in
copulatory behavior (Blockey, 1981).
Absence of adequate sexual preparation prior to semen collection reduces the libido and
courtship behaviour followed by deficient vigor of the ejaculatory reflex. Whereas, sexual
preparation will increase the number of sperm ejaculated (Amann, 1990).
Almquist (1973) using Angus, Hereford and Holstein bulls to determine the effects of sexual
preparation by false mounting on sperm output, semen traits and sexual activity demonstrated that
giving 3 false mounts rather than no false mounts before semen collection increased sperm output by
about 50% in first ejaculates for both beef and dairy bulls. He also found that changes of dummy and
semen collection location were commonly required to stimulate many of the beef bulls and to
maintain their sexual interest during sexual preparation.
Libido or the willingness and eagerness of a bull to mount and to complete service may be
assessed by several indices viz. reaction time, number of ejaculations in a constant period of time
and intensity of thrust during ejaculation. The stronger intensity of thrust produce ejaculates with
more volume, sperm concentration per unit of volume and better motility. Though apparently a
shorter reaction time was found to yield better quality semen, such influences were significant only
for volume and individual motility. Besides genetic factors, libido is affected by nutrition, ambient
temperature or season, age, systemic disease and abnormalities or pathology of locomotor system.

Collection Interval
Shorter intervals between collection days caused ejaculate volumes to decrease slightly, sperm
concentration to decrease markedly, and sperm output per unit of time to increase considerably
(Hafs, 1959). Amann and Almquist (1976) reported that 3.5 times more motile spermatozoa could be
collected from 1 to 2 year old Holstein bulls when six ejaculates per week were collected as
compared with one ejaculate per week. However, 40% more time was required to obtain an ejaculate
on the six ejaculates per week schedule. They concluded that with appropriate sexual preparation,
similar spermatozoal harvests could be achieved by collecting two to three ejaculates every 3 to 4
days.

Semen Collection Management

Proper and clean harvesting of the ejaculates is the most important aspect of any artificial
insemination programme and therefore proper management of the entire collection process is
critical. Many methods of semen collection like artificial vagina (AV), electro-ejaculation, massage
technique have been used. The most popular and accepted procedure is AV method and bulls enjoys
the charms of almost natural mating. The AV should be properly assembled maintaining sterile
conditions as far as possible. The internal temperature of AV should be maintained at 41-42oC for
young and 42-45oC for adults. Some older bulls prefer to ejaculate at 2-3oC higher temperature. The
design of the artificial vagina has changed slightly over the years to reduce sperm losses and the risk
of damaging the penis. Small sized (20-25 cm) AVs are now commonly used for collection of semen
from buffalo bulls and young cow bull whereas 30 cm long AVs are used for bulls having long sized
penis so that the ejaculates are directly collected in the collection tube maintained at about 35oC
without exposure to the higher temperature of the rubber lining.
There are several non-negotiable responsibilities that must be adhered to accomplish the goal of
obtaining a high quality ejaculate, thus maximizing the sperm harvested from the bull. These
responsibilities include:

(i) Trained personal: Semen should be collected by a trained collector using properly
prepared AV. While bulls mount on dummy, semen collector should give support to bulls by
shoulder and divert the penis holding prepucial sheath to 45oC angle towards AV for allowing
the bull to give thrust into it. Semen collector should wear disposable hand gloves to avoid
 contamination and getting infected from zoonotic diseases. After end of collection properly
wash hands, face (if required) and other exposed part by savlon or mild soap solution.

(ii) Employee Safety: Bulls are inherently dangerous. Employees must observe much cautious
during handling of the bull in collection yard. Semen collector should wear white/light coloured
apron and gum boot to avoid injury.

(iii) Bull Safety: Protection from injury during the collection process is of the utmost
importance. A collection area with non-slip flooring is needed for safe movement of bulls
during teasing and semen collection. Alternatively a rubber/ coir mat may be used. Ejaculate
quality and quantity may be negatively affected by improper footing.

(iv) Disease Transmission: Bulls should be health tested before entering isolation facilities, during
the isolation period, and during residency at the AI center. Precautionary care to minimize the
exchange of bodily fluids between bulls must be exercised. Mounting of bulls on a common
teaser is a source of contamination. Wash the back and rear quarter of the teaser with a
disinfectant between bulls or bull aprons made of soft rexin cloth (approx. 65 x 62 cm) should
be used during semen collection. A separate AV should be used for each collection attempt.

(v) Identification of Ejaculates: Accurate ejaculate identification can be achieved only if the bull is
properly identified. Any ejaculate leaving the semen collection arena without unequivocally
identifying the source should be discarded. Avoid exposure of direct sun light/UV rays to
collected semen. Use of advanced identification systems such as ‘SMILE’ may be of immense
use if budget permits.

(vi) Mount Animal: Dummy males are commonly used in collection facilities for sexual stimulation.
To achieve sexual stimulation in the shortest amount of time one can use a combination of
mounts, familiar mounts in different location, or new mounts in different locations. Mounts
should be selected based on size, temperament, and disease status (Miller, 1992).

(vii) Proper scheduling: Frequency of semen collection should be decided based on the age
and body weight of the bull. Very young bulls should be used for collection once in 15 days
whereas biweekly double ejaculates from bulls aged 2 to 7 years and weekly from aged more
than 7 years can be safely collected with out any deleterious effect on the libido and semen
quality.
Evaluation of semen pre and post freezing
Irreversible changes in the semen leading to sperm aging begins soon after the spermatozoa are
collected. Therefore routine evaluation methods for assessing the quality of semen should be
completed as early as possible. Inspite of many evaluation methods developed during the last decade
only a few are used for routine evaluation particularly at semen freezing stations so that semen is
processed for freezing and stored at ultra low temperature within the shortest possible time.
The quality parameters that are assessed routinely in neat semen are
visual appearance, volume, sperm concentration and total number of
spermatozoa, sperm motility, and sperm vitality. Furthermore, differential count
with respect to sperm morphology, acrosomal morphology and assessment of
presence of debris and other cell types in semen are also performed at regular
interval to assess the fertilizing capacity of the spermatozoa produced by
individual bulls.
Sperm concentration and initial motility is determined on a small aliquot
of the ejaculate. This measurement is important because it ultimately
determines the number of sperm in each straw. The cumbersome methods for
determination of sperm concentrations have been simplified over the years and
now in almost all the laboratories spermatozoa concentration is measured by
the photo electric colorimetric instruments manufactured by some leading
firms( IMV, France and Minitube, Germany) dealing in AI instruments.
After freezing and thawing, only viable spermatozoa carrying intact genetic information are
potentially fertile and therefore, most of the methods used so far focus on sperm viability and DNA
integrity. Fertility of sperm is the ultimate test of sperm quality. Often it is not
possible to measure fertility, so many tests of semen quality in addition to
motility and morphology, such as the hypoosmotic swelling test, mucous or gel
penetration, and integrity of the DNA have been correlated with fertility
(Graham, 1978; Saacke, 1981; Foote, 1999).The parameters which are currently used for
assessment of spermatozoa quality are given below.

Motility
Due to the simplicity of the evaluation technique motility is probably the most often used criterion
for routine semen evaluation. The technique is simple and motility is assessed by light microscopy
under phase contrast optics which also gives an assessment of the morphological status of the
spermatozoa. Spermatozoa are graded in to several types based on the patterns of motility ( straight,
curvilinear or oscillatory) and speed of progression. However, since the estimates are largely
subjective, the accuracy of this depends on the ability of the technician. Although some researchers
have observed a significant relationship between subjective motility and field fertility (Kjaestad et
al., 1993) others found inconsistent results (Stalhammar et al., 1994). Computer assisted semen
analysis (CASA) is comparatively a new but reliable approach for motility and morphological
estimations since it operates on objective assessment based on the kinetic properties of individual
spermatozoa and is free from human error provided the instrument has been properly calibrated and
validated. Estimates of motility and movement characteristics of sperms by CASA have been
positively correlated with in vivo fertility ( Budworth et al., 1988 ).

Energy estimation
ATP is the ultimate source of energy to the live cells. The concentration of ATP in semen has been
shown to be related to the number of motile spermatozoa. Measurement of ATP concentration in
semen can indirectly measure the number of viable spermatozoa and thus may provide an objective
method of estimating sperm viability than measuring motility by visual methods (Januskauskas and
Rodriguez-Martinez,1995). However, results concerning correlations between sperm ATP
concentrations and fertility have been contradictory (Soderquist, et al 1991, Januskauskas et al,
1996).

Sperm Viability
Intactness of the plasma membrane is essential for the spermatozoa to perform its physiological
functions. Many supravital staining dyes such as trypan blue, eosin, aniline blue, and some other are
widely used for differential live/dead staining of spermatozoa. Due to exclusion of these dyes by the
intact plasma membrane these staining techniques are used to differentiate between live and dead
cells, the live cells on incubation with the dyes remain unstained. However, these dyes are
commonly used for the viability test of freshly ejaculated spermatozoa since glycerol, the commonly
used cryoprotectant interferes with the staining. The use of supravital dyes and light microscopy is
being taken over by the fluorescent dyes such as Propidium iodide and Ethidium bromide to assess
the plasma membrane integrity of frozen thawed spermatozoa. These dyes bind to DNA as well as to
RNA by intercalating between the bases. These dyes can be excited with mercury or xenon-arc
lamps and are suitable for fluorescence microscopy and confocal laser scanning microscopy. SYBR-
14, a new membrane permeant nuclear stain for living cells (Garner and Johnson,1995) has been
tested in combination with propidium iodide to assess bovine sperm viability. This combination of
dyes has several advantages over the above-mentioned dyes, among which is higher speed of
labeling cells, and higher stability while labeled. In addition, both dyes fluoresce at excitation with
visible light, thus avoiding harmful effects of UV exposure. Under illumination, SYBR-14 loaded
viable sperm cells fluoresce bright green, whilst damaged cells take-up propidium and fluoresce red.

Sperm chromatin structure assay (SCSA)

Morphological abnormalities in the spermatozoa are either of genetic origin particularly observed in
some breeds or due to defective spermatogenesis as a result of insult to the spermatogenic cells by
thermal or other toxicants. Classification of normal and abnormal morphological shapes of
spermatozoa assessed by visual examination under light microscopy are inconsistent with abnormal
sperm chromatin structure (Ballachey etal,1988)Whereas, the abnormal sperm chromatin structure
has been found significantly correlated with abnormal bovine sperm nuclei (Sailer etal, 1996). SCSA
evaluates the denaturation of Sperm DNA using flow cytometry. Suspension of Acridine orange
stained spermatozoa are run through flow cytometer and excited with blue laser light. The Acridine
orange intercalated into double stranded DNA fluoresce greed whereas tagged with fluoresce red. It
has been shown that structural changes in the chromatin of spermatozoa could be detected as early as
3 days after thermal insult to the scrotum, whereas light microscopy could not reveal the
abnormalities until 11 days. Thus SCSA could be a valuable complement to the routinely practiced
methods of morphological evaluation of spermatozoa by light microscopy.

Hypoosmotic swelling test (HOST)

The functional integrity of plasma membrane of the spermatozoa can also be evaluated by simple
test based on the water influx into the cytosol when exposed to hypoosmotic solutions. On exposure
to hypoosmotic solution (150 mOsm/Kg) water enters the cytosol of plasma membrane intact (live)
spermatozoa till the equilibrium is established. Attainment of the equilibrium is visible by swelling
of the sperm which is observed microscopically. Dead spermatozoa do not show these changes as
plasma membrane is not able to regulate the transport of water and remain osmotically inactive.
Thus hypoosmotic swelling test may be useful in evaluation of functional integrity of sperm cells
during freezing and thawing( Revell and Mrode, 1994.) Unfortunately this test is not always
correlated with the fertility trials and the results obtained varied from studies and methods used.

Zona binding and sperm penetration assay

Fertilization is the ultimate test of semen quality. Spermatozoa are required to pass through a
sequence of events before the male and female pronuclei combine together and result in the
formation of zygote. Sperm egg interaction can be used as a predictive test for assessing the
fertilizing potential of spermatozoa.Two types of binding assays viz. zona binding assay (ZBA) and
hemizona binding assay (HZA) can be used to predict the binding capacity of spermatozoa of
individual bulls. In ZBA spermatozoa are incubated with the intact homologous oocytes ( Fazeli et
al., 1993) whereas, in HZA (Fazeli et al., 1997) bisected halves of homologous oocytes are cultured
with the spermatozoa of bulls of known fertility and test sample. Thus it enables a comparison of
sperm binding between fertile controls and test bulls.
Species specific receptors are present on the zona pellucida which allows selective binding of
spermatozoa from homologous species and prevents the sperm egg encounter between species.
However, onces the zona barrier is removed sperm from different species can penetrate through the
of heterologous oocyte. In view of this, zona free hamster egg penetration assay was developed for
in vitro assessment of predictive fertilizing capacity of spermatozoa from heterologous species.
However, these in vitro test methods have so far been used on limited number of animals, are
expensive, require expertise and are not consistently reproducible.
Preservation
Extenders
After the advent of AI the focal point of research was to find out the methods to
store the semen for extended period of time (long enough for shipment and use
in the field). This lead to the development of a yolk-phosphate semen extender
by Phillips and Lardy in 1940. Salisbury et al., (1941) later on improved the
media by buffering the egg yolk with sodium citrate. Both these diluters were
extensively used to store the semen at refrigerator temperature for 72 to 96 h.
However greatest boost to the technique of semen preservation was made by
the discovery of Glycerol as the cryoprotective agent (Polge et al.,1949).
Subsequently Davis et al. (1963) found that Tris (hydroxy methyl amino methane)-
yolk-glycerol gives better buffering to the diluted semen as reflected by
maintenance of 56% motility for 4 days compared to 35% in egg yolk citrate
glycerol dilutor. Since than continuous efforts are being made to develop new
dilutors and modify the technique of freezing at low temperature. Over the
years, many dilutors and a variety of cryoprotectants have been tested,
however, Tris and glycerol have been recognized as dilutor and cryoprotectant
of choice for bovine semen Freezing. Earlier, the glycerol addition was made to
the buffered semen at 4oC in view of toxic effect of glycerol to the spermatozoa
at ambient temperature in presence of egg yolk. However, Foote in 1970
discovered that inclusion of glycerol in Tris dilutor at room temperature is as
effective as at 5oC. This discovery leads to simplification of the technique of
semen freezing and helped to avoid possibilities of temperature variation due to
frequent handling of semen during processing for freezing. Now single step
dilution with Tris- glycerol- egg yolk dilutor is a standard practice all over the
world.

Reactive oxygen species scavangers

The semen dilutors have been fortified with certain additives from time to time
by inclusion of substances which help to maintain sperm plasma membrane
stabilization and regulate the generation of reactive oxygen species during
processing and storage . Cryopreservation is associated with oxidative stress due to
production of reactive oxygen species (ROS) which leads to lipid peroxidation of sperm membrane.
Naturally occurring anti-oxidant viz. vitamin C, α-tocopherol, Super oxide dismutase exert a
protective effect on the plasma membrane of spermatozoa (Askari et al., 1994; O’Flaherty et al.,
1997). Other substances viz. thiol group of chemicals (Bilodeau et al., 2001) e.g. cysteine, 2-
mercaptoethanol and glutathione etc and like wise BHT, selenium, manganese and zinc etc. have
also been found to have anti oxidant properties and been used in cryopreservation medium for semen
.

Protein source
Glycerol and low-density lipoproteins from egg yolk are the commonly used cryoprotectants in
cryopreservation of bull semen since many years. But in the last few year’s new diluents with
lecithin based cryoprotective components were introduced into practice. Because these extenders
work without egg yolk, they have some advantages in laboratory handling and in the defense of risks
existing for the quality of cryopreserved spermatozoa by the biological and bacteriological
properties of egg yolk. Biociphos Plus and BIOXCELL (IMV Technologies, L’Aigle, French), and
AndroMed (Minitüb Abfüll- and Labortechnik GmbH & Co. KG, Tiefenbach, Germany) without
animal protein source have been tested for cryopreservation of bull semen and it has been seen that
in the field trials egg-yolk free diluents BIOXCELL or AndroMed caused similar non-return rates in
comparison with the egg-yolk containing extender Triladyl if 15 millions spermatozoa per
insemination were used (Nehring and Rothe,2003).

Freezing Methodology:
Till few years back semen was frozen in 0.5/0.25 ml straws in wide mouth liquid nitrogen
containers. Horizontally spread and cooled straws (4-5oC) were vapour frozen at a height of 4 cm
above the liquid nitrogen level. Freezing for 8-10 minutes could lower the temperature of semen to
about -140oC before being finally plunged to liquid nitrogen. Certain requirement based
modifications in duration of vapour freezing were common in different laboratories. The biggest
drawback of this methodology was the variation in the freezing rate between different batches due to
change in the level of nitrogen and also because of ambient temperature of laboratory. Straw to straw
variation was also evident in the straws of same batch. With the use of bio freezer for semen freezing
these shortcomings have now been eliminated. Even, it is possible to store bull specific freezing
curves and thereby it is possible to preserve the semen from high pedigreed bulls of low freezability.
Recently a new concept of freezing “batch freezing” has been reported by Amir ARAV et al.,
(2003) by successful freezing of a large volume of semen in 12 ml tube. Once the bull is found to be
proven, the test tube is thawed and semen will then be refrozen in regular mini (0.25 ml) straws.
This technology devised by Arav (1999) is named as “Multi-thermal gradient MTGR, and the
semen in the test tube is moved at a constant velocity (V) through a linear temperature gradient (G)
so the cooling rate (G x V) and ice front propagation are precisely controlled. This technique once
standardized and equivocally accepted will help in minimizing storage space for semen and
expenditures on liquid nitrogen.

References
1. Budworth PR, Amann RP, Chapman PL.1988. Relationship between computerized measurements of
motion of frozen-thawed bull sperm and fertility. Journal of Andrology. 9 : 41-54.
2. Almquist, JO. 1973. Effects of sexual preparation on sperm output, semen characteristics, and sexual
activity of beef bulls with a comparison to dairy bulls. J.Anim. Sci. 36: 331.
3. Amann, RP. 1990. Management of bulls to maximize sperm output. in Proc.13th Tech. Conf. on A.I.
and Repro., NAAB. Milwaukee, Wisconsin. Pages 84-91
4. Amann, RP and Almquist JO. 1976. Bull management to maximize sperm output. in Proc. 6th Tech.
Conf. on A.I. and Repro., NAAB. Milwaukee, Wisconsin. Pages 1-10
5. Amir ARAV, Yoel ZERON, Haim SHTURMAN, Haim GACITUA. 2002. Successful pregnancies in
cows following double freezing of a large volume of semen. Reprod. Nutr. Dev. 42 : 583–586.
6. Arav A., 1999. Device and methods for multigradient directional cooling and warming of biological
samples, US Patent . 5,873,254.
7. Askari HA, Check JH, Peymer N and Bollendorf A. 1994. Effect of natural anti-oxidants tocopherol
and ascorbic acids in maintenance of sperm activity during freeze-thaw process. Arch Androl. 33: 11-
15.
8. Ballachey BE, Evenson DP, Saacke RG. 1988. The sperm chromatin structure assay: Relationship
with alternate tests of semen quality and heterospermic performance of bulls. Journal of Andrology.
9: 109-115.
9. Bearden HJ, Fuquay JW. 1997. Applied Animal Reproduction. 1997. 4th ed. Upper Saddle River,
New Jersey, Prentice Hall, Inc.: 42-146.
10. Bilodeau JF, Blanchette S, Gagnon C and Sirard MA. 2001. Thiols prevents H2O2- mediated loss of
sperm motility in cryopreserved bull semen. Theriogenology 56: 275-286.
11. Blockey MADB. 1981. Modification of a serving capacity test for beef bulls. Appl.Anim. Ethol. 7:
321-336.
12. Davis IS, Bratton RW and Foote R H. 1963. Livability of bovine spermatozoa at 5, −25, and −85oC
in tris-buffered and citrate buffered yolk-glycerol extenders. J. Dairy Sci. 46:333–336.
13. Fazeli AR, Zhang BR, Steenweg W, Larsson B, Bevers MM, van den Broek J, Rodriguez-Martinez
H, Colenbrander B. 1997. Relationship between sperm-zona pellucida binding assays and the 56- day
nonreturn rate of cattle inseminated with frozen thawed bull semen . Theriogenology 48:853-863.
14. Fazeli AR, Steenweg W, Bevers MM, de Loos FAM, van den Broek J, Colenbrander B. 1993.
Development of a sperm zona pellucida binding assay for bull semen. Vet Rec 132:14-16.
15. Foote RH, Chen Y, Brockett CC. 1993. Fertility of bull spematozoa frozen in whole milk extender
with trehalose, taurine, or blood serum. J Dairy Sci. 76: 1908-1913.
16. Foote RH. 1982. Cryopreservation of spematozoa and artificial insemination: past, present, and
future. J Androl. 3: 85-1 00.
17. Foote RH. 1999. Artificial insemination from its origins up to today.In: V Russo, S Dall ’Olio, and L
Fontanesi (ed.) Proc. of the Spallanzani Int. Symp., Reggio Emilia, Italy. pp 23–67.
18. Garner DL, Johnson LA. 1986. Viability assessment of mammalian sperm using SYBR-14 and
propidium iodide . Biology of Reproduction. 34: 127-138.
19. Hafs HD, Hoyt R S and Bratton RW. 1959. Libido, sperm characteristics, sperm output, and fertility
of mature dairy bulls ejaculated daily or weekly for 32 weeks. J. Dairy Sci. 42: 626-636.
20. Januskauskas A, Rodriguez-Martinez H. 1995. Assessment of sperm viability by measurement of
ATP, membrane integrity and motility in frozen/thawed bull semen. Acta vet scand; 36:571-574.
21. Januskauskas A, Haard MG, Haard MGH, Söderquist L, Lundeheim N, Rodriguez-Martinez H. 1996.
Estimation of sperm viability in frozen-thawed semen from Swedish AI bulls . Journal of Veterinary
Medicine series A 43:. 281-87.
22. Johnson LV, Walsh ML, Chen LB. 1980. Localization of Mitochondria in Living Cells with
Rhodamine 123 . Proceedings of National Academy of Sciences USA. Vol. 77. P. 990.
23. Kjaestad H, Ropstad E, Andersen Berg K. 1993. Evaluation of spermatological parameters used to
predict the fertility of frozen bull semen. Acta vet scand; 34: 299-303.
24. Miller R. 1992. Importance of the mount animal in semen collection. in Proc. 14th Tech. Conf. on
A.I. and Repro., NAAB. Milwaukee, Wisconsin. Pages 98-99
25. Nehring H, Rothe L. 2003. Insemination of cryopreserved bull semen portions with reduced sperm
numbers after dilution with two egg yolk free extenders. Proceed. European A.I. Vets Meeting (Cattle
session, 14 – 23) Budapest (Hungary), 8 – 11 October, 2003
26. O’Flaherty C, Beconi M and Beorlegui N. 1997. Effect of natural anti-oxidants superoxide dismutase
and hydrogen peroxide on capacitation of frozen thawed bull spermatozoa. Andrologia 29: 269-275.
27. Pennington D. 1990. Management of the semen collection arena. in Proc.13th Tech. Conf. on A.I. and
Repro., NAAB. Milwaukee, Wisconsin. Pages 92-94.
28. Phillips PH, and Lardy HA. 1940. A yolk-buffer pabulum for the preservation of bull semen. J. Dairy
Sci. 23:399–404.
29. Polge C, Smith AU and Parkes AS. 1949. Revival of spermatozoa after vitrification and dehydration
at low temperatures. Nature (Lond.) 164:666.
30. Revell SG and Mrode RA. 1994. An osmotic resistance test for bovine semen. Animal Reproduction
Science. 36: 77-86.
31. Saacke R G. 1981. Components of semen quality. J. Anim. Sci. (Suppl. 2) 55:1–13.
32. Sailer BL, Jost LK, Evenson DP. 1996. Bull sperm head morphology related to abnormal chromatin
structure and fertility. Cytometry. 24: 167-173.
33. Salisbury GW, Fuller HK and Willett EL. 1941. Preservation of bovine spermatozoa in yolk-citrate
diluent and field results from its use. J. Dairy Sci. 24:905–910.
34. Smith AU, Polge C. 1950a. Storage of bull spematozoa at low temperatures. Vet Rec. 62: 7 15.
35. Smith AU, Polge, C. 1950b. Survival of spematozoa at low temperatures. Nature; 166: 668.
36. Söderquist L, Rodriguez-Martinez H, Janson L. 1991. Post-thaw motility, ATP content and
cytochrome C oxidase activity of A.I. bull spermatozoa in relation to fertility. Journal of Veterinary
Medicine. 38: 165-174.
37. Stalhammar EM, Janson L, Philipsson J. 1994. The impact of sperm motility on non-retun rate in
preselected dairy bulls. Reprod Nutr Dev; 34:37-45.
38. Stewart DL. 1951. Storage of bull spematozoa at low ternperatures. Vet Rec 63165-66.