Will explain this in detail. This page is just for remembering for the time being.

Structure

From Wikipedia, the free encyclopedia

Composition of Sperm membrane

It contains a wide variety of biological molecules, primarily proteins and lipids, which are involved in a vast array of cellular processes, and also serves as the attachment point for both the intracellular cytoskeleton and, if present, the cell wall

Function
All eukaryotic cells have a semi-permeable lipid bilayer known as the cell membrane or plasma membrane or "phospholipid bilayer" (Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell, 4th ed.. ISBN 0-8153-3218-1. ). Sperm plasma membrane performs many functions such as securing the cytoskeleton, maintaining chemical gradient of ions because of its semi permeable features between the cell and its immediate environment transport of glucose and fructose from extracellular environment into the sperm through specific plasma membrane proteins.

Effective liquid and cryo preservation of the emu semen is necessary for developing an AI programmne ( Malecki and Martin _Emu Farming 2000_____). Presently emu spermatozoa can be stored undiluted or diluted only upto 6 hours at 20 C or at 4 C respectively ( Malecki et al 2005b). This enables sperms to be used only within a farm. In order to harness full potential of selective gene storage/transfer it is vital to achieve an effective short and long-term storage of semen. However, cryopreservation of emu semen has not given encouraging results till date. ( Malecki and Martin 2006). Different cryopreservation methods have been tried but none have given encouraging results, as there is a considerable loss of sperm function. The fertilizing ability is lost by 87% while motility by 63% (Malecki and Martin 2006) indicating that sperms are vulnerable to washing, dilution temperature and osmotic changes involved in the sperm storage (P.F.N. Silva , B.M. Gadella 2006). It is pertinent for the membrane to preserve its integrity through the entire freeze thaw cycle so as to retain integrity and fertilizing capacity. However empirical cryopreservation approaches have not achieved acceptable levels of fertility in most domestic animal species (J.E. Park and J.K. Graham 1992). During diverse steps of sperm storage, the sperm plasma membrane and plasma membrane of its organelles are vulnerable to damage ((P.F.N. Silva , B.M. Gadella). This damage needs to be both identified and measured to accurately predict the fertilizing capacity of the spermatozoa for its functional competent membranes, organelles and an intact haploid genome (PFN. Silva, B.M Gadella 2006). Many laboratory assays have been developed over the years to allow in accurate prediction of this fertilizing capacity but encouraging results have not been obtained because of the complex disturbances that can occur to the spermatozoa plasma membrane (J. K. Graham 1992), organelles and genome (PFN. Silva, B.M Gadella 2006). This is because each assay evaluates different characteristics of the spermatozoa e.g. visual examination or computer assisted methods evaluate percentage of motility, exclusion and inclusion dyes assess morphology and viability whereas fluorescent probes evaluate organelle 2

integrity of acrosomes and mitochondrial. Hence as explained a single test is not able to assess all the attributes that a sperm is required to posses for a successful fertilization. Moreover each assay method has inherent advantages and limitations and may introduce specific biases into the experiment; thus, different applications often call for different approaches. Hence more than one test needs to be carried out to in an attempt to effectively predict the fertilization capacity. Malecki and Martin _Emu Farming 2000__, or ( Malecki and Martin _Emu Farming 2000_____) have employed different tests such as HOST to determine sperm membrane integrity, staining techniques with exclusion and inclusion dyes (e.g. nigrosin eosin, combinations of Calcenin-AM, Propidium Iodide and SYBR-14) to estimate viability of emu spermatozoa , evaluating effect of season, dilutions and storage on different parameters of semen ejaculate (such as volume, concentration, number of spermatozoa, live and abnormal spermatozoa, measuring ratio of unsaturated to saturated fatty acids, composition of seminal plasma and sperm egg interaction assay etc, . It can be appreciated that presently employed assays for evaluation of emu spermatozoa do not measure attributes like membrane integrity of of organelles like mitochondria and acrosome which have been shown to be highly correlated with the fertilizing ability multiple attributes and hence are not sufficient to correlate with effectively predicting fertilizing capacity of the sperm. For example a sperm sample classified as fertile according to the presently employed tests by Malecki and Martin but may have a damaged acrosome or a damaged mitochondria that may prevent it from fertilizing the egg thus giving erroneous results (as the sperm sample would be classified as infertile by an assay that measures multiple sperm attributes). In consequence there is a need for using tests that can simultaneously evaluate multiple sperm attributes such cell viability, acrosomal and mitochondrial integrity measuring LDH (Lactate dehydrogenase C4 iso-enzyme (LDH-C4) is involved in
the energy metabolism of spermatozoa. Plasma membrane LDH may participate in the production of lactate to obtain intracellular reducing equivalents to be used by sperm oxidase for in vitro sperm capacitation , O'FLAHERTY C. M. ; BEORLEGUI N. B. ; BECONI M. T. Lactate dehydrogenase-C4 is involved in heparinand NADH-dependent bovine sperm capacitation. Andrologia ISSN 0303-4569 CODEN ANDRDQ

Source / Source 2002, vol. 34, no2, pp. 91-97 (39 ref.)

and GOT ( Mitochondrial and Cytoplasmic and

involved in the cell in the formation of amino acids) and functions apart from chromosomal integrity so as to effectively predict the percentage of fertile sperms in a semen population (Surrai and Ionov 1981, Surai 1983a). Nevertheless the tests should be extremely efficient, objective, repeatable, simple, robust, and relatively inexpensive per unit (G. J. Wishart 2007, J. K. Graham 2001) and should have the ability to be performed immediately and quickly after thawing the spermatozoa sample. There are many laboratory assays that have been developed by scientists that can be used for testing plasma membrane and organelle integrity so as to accurately predict the fertilizing capacity of the semen sample (J. K. Graham 2001). The ones varying from the most simple to the most sophisticated ones are being discussed here as a part of literature review of the thesis.

TESTS FOR PLASMA MEMBRANE AND ORGANELLE INTEGRITY BY EVALUATING: A) Plasma Membrane
In case Seminal Plasma membrane is damaged the sperm is considered deteriorated (dead) and in vivo looses it fertilizing capacity ( PFN Silva, B.M Gadella 2006). However for artificial insemination it is essential to determine plasma membrane integrity of liquid or cryo-stored semen sperm. This can be done by calculating percentage of viable sperm in a semen sample which is defined as the percentage of sperm that possess intact plasma membranes and can therefore keep large molecules (stains) out of the cell, can be evaluated using a variety of stains.: SEM/TEM: Can be used to determine sperm membrane integrity Advantages: Individual spermatozoa and its organelles can be studied in detail (Froman and Kirby 2005/ G.J. Wishart 2007) Disadvantages: These techniques are not suitable for routine sperm quality assay, are time consuming, expensive and subjective.

STAINING PROTOCOLS FOR EXCLUSION AND INCLUSION DYES FOR MEMBRANE INTEGRITY: i. Membrane Impermeable Non Fluorescent Dyes: Dried semen smear (and hence can only be tested on spermatozoa that are not use for AI) have been used to stain sperms with exclusion dye (such as eosin, aniline blue or trypan blue) in conjunction with a background stain (such as nigrosin) under various concentrations and exposure times (Bjorndahl, Lars; et.al, Assessment of Sperm Quality, James K. Graham, PhD, 302 2001 / Vol. 47 / AAEP PROCEEDINGS) to simultaneously determine sperm morphology and sperm membrane integrity. This test uses the principle that only damaged sperm membranes allows impermeable dyes to pass through it thereby organelles of only those sperms would be stained by membrane impermeable dyes (e.g. eosin) that have a damaged plasma membrane. However this test measure the structural but functional integrity of the plasma membrane (J.M. Vazquez , et al 1997 Theriogenology). ii. Membrane Impermeable Fluorescent dyes/probes (also know as Cell Membrane Impermeant dyes) : Provide greater contrast between live and dead cells and work on the same principle as of Membrane Impermeable Non Fluorescent Dyes but require a microscope with fluorescent capabilities for analysing spermatozoa. A wide variety of such probes are available for testing sperm cells each having an exclusive excitation (ex) and emission (em) properties as given below

Sr. No

Fluorescent Dyes/Probes

Ex/Em (in nm wavelength) 358/488 488/515 488 and 568/> 620 for both 647/670 for both 360/453nm.

Reference Hong CY et al 1988, Mac Laughlin et al 1988 Harrison RA et al 1996 Pintado B et al 2000, Garner DL et al 1986 Cheng FP et al 1996 Huagland RP 2004 J. Ch. Meyer1 and H. Grundmann *

1) 1 Hoechst 33258 2) 2 YoPro-1 3) 3 Propidium Iodide (PI) or Ethedium Homodimer-1 (EH 1) 4) 4 ToPro 3 and TOTO 5) 5 Diamidino-Phenylindole (DAPI)

* Fluorometric determination of DNA in epidermis and cultured fibroblasts, using 4-6-diamidino-2-phenylindole (DAPI) Journal Archives of Dermatological Research Volume 276, Number 1 / January, 1984;52-56.

iii) Acetylated Membrane (AM) loading Dyes (also known as Cell membrane permeant dyes) : AM loading dyes are used to study membrane integrity of live sperms e.g. Fluorescin diacetate and Carboxy (methyl) derivatives (green fluorescent) (Garner DL et al 1986, Donoghue A M 1995). SYBR-14 (green fluorescent) and Calcein AM (green fluorescent) is a recent addition of AM loading dyes introduced for use in evaluation of metabolically-intact and membrane-intact cells. These dyes possess acetyl moieties and are amphipathic (possess one polar and one non- polar end) in nature as a result of which they can penetrate the intact/living sperm plasma membrane. Once in the intact cell cytoplasm these AM probes are deacylated by intracellular esterases (de-esterification) and henceforth cannot diffuse back over the intact plasma membrane. These entrapped deacylated probes stain the sperm nucleus as they have a weak affinity for DNA thus staining nucleus of metabolically-intact, membrane-intact cells only. (Wishart G.J. 2007 P.F.N Silva et al 2006). However these AM probes can easily leak out of the damaged cells having a damaged plasma membrane. This characteristic of AM loading dyes gives an opportunity to play with different combinations of fluorescent and AM loading dyes for example Propidium Iodide or Ethidium Homodimer-1 (red fluorescent) can be used with SBYR-14 (green fluorescent) ( Chalah and Billard 1998) or Carboxyfluorescein Diacetate (green fluorescent) (Bayyari et al 1990) to detect metabolically-intact, membrane-intact sperm cells. These novel fluorescence combinations are available commercially as LIVE/DEAD Sperm Viability Kit (Molecular Probes, Invitrogen detection technologies) where they are being used for analyzing the viability and fertilizing potential of sperms. One Sperm Viability Kits employs a combination of SYBR 14 (a membrane-permeant nucleic acid dye) and Propidium Iodide (a dead-cell stain) to label live sperms as green fluorescence and dead ones as red-orange fluorescence respectively. Similarly another kit use uses a combination membrane-permeant nucleic acid dye (Calcein AM which has green fluorescence in live sperms) and a dead-cell stain (Ethedium Homodimer -1 which has bright red fluorescence in 6

dead cells) to differentiate live and dead cells with visible-light excitation (Molecular Probes, Invitrogen detection technologies). This differentiation can be done either manually or through flow cytometry as was done for turkey spermatozoa (Donghue et al., 1996). Manual assessment is time consuming and inefficient to flow cytometric assessment. Recent developments in flow cytometry10,11 make viability assessments rapidly and inexpensively as it measures multiple sperm attributes from thousands (50,000) of spermatozoa in less than 1 minute without the need to perform any laborious processing such as dilution or centrifugation (J. K. Graham 2001)

10. Graham JK, Kunze E, Hammerstedt RH. Analysis of sperm viability, acrosomal integrity, and mitochondrial function using flow cytometry. Biol Reprod 1990;43:5564. 11. Garner DL, Johnson LA, Yue ST, et al. Dual staining of bovine sperm viability using SYBR-14 and propidium iodide. J Androl 1994;15:620629.

Hypo Osmotic Swelling Test (HOST): It is a sensitive and reproducible test to assess the functional integrity of sperm membrane as shown by Vaquez , J. M.;et.al 1997 (Theriogenology 47:913-922, 1997). It was originally developed to test water permeability of sperm membrane based on the principle that live sperms normally actively exclude water by osmo-regularion, but if suspended in hypoosmotic solutions this capacity is overcome and they will undergo reversible swelling of the tail membrane in case there is involvement of the principal piece and not the mid piece as reported by J.M. Cummins for human spermatozoa ( in Gametes The Spermatozoon Edited J.G Grudzinskas and J.L. Yovich < Cambridhge University Press pg 84). Vette and al. described that swelling of sperm tail is faster in fast sperms than in slow sperms [7]. Correlation between HOST and fertilization is not unambiguous as contrasting results have been reported by various different authors such as Hauser et al. [9] who found a correlation between the proportion of reacted sperms and fertilization while Biljan et al. [10] did not. Nowdays HOST is commonly used for selection of viable but immotile spermatozoa used for ICSI (Experiences with hypoosmotic 7

swelling test Sz. Mtys1, I. Balogh2, K. Rajczy1, A. Bernard1, Gy. Papp3, I. Gti1, S.G. Kaali1 http://www.krio.hu/downloads/hypoosmotic.pdf.). Malecki and Martin 2005 (Reproductive Technologies for ratite Farming) have used this to evaluate sperm membrane integrity after storage and for detection of inferior ejaculates. In the case of frozenthawed spermatozoa the freeze-thaw process disrupts the outer and inner membrane, resulting in loss of interaction contents, but this does not necessarily affect the tail region [11]. Intact microstructure is required, because spermatozoa do not solve only as DNA sources. Moreover it should be noted that the reason of immotility strongly affects the fertilization and later embryonic development (Experiences with hypoosmotic swelling test Sz. Mtys1, I. Balogh2, K. Rajczy1, A. Bernard1, Gy. Papp3, I. Gti1, S.G. Kaali1
http://www.krio.hu/downloads/hypoosmotic.pdf.).

[7] Vette M, Petzoldt L, Engel S, Roder B, Reichmann G. The relation between sperm motility and the content of unsaturated fatty acids as well as the osmotic behaviour of human spermatozoa. Andrologia 1985 Sep; 17(5): 476-84. [9] Hauser R, Yavetz H, Paz GF, Honozuli ZT, Amit A, Lessing JB, Peyser MR, Yogev L. The predictive fertilization value of the hypoosmotic swelling test (HOST) for fresh and cryopreserved sperm. J Assist Reprod Genet 1992 Jun; 9(3): 265-70. [10] Biljan MM, Taylor CT, Manesse PR, Joughin EC, Kingsland CR, Lewis-Jones DI. Evaluation of different sperm function tests as screening methods for male fertilization potential - the value of the sperm migration test. Fertil Steril 1994 Sep; 62(3): 591-98. [11] Cross NL. and Hanks SE. Effects of cryopreservation on human sperm acrosomes. Hum Reprod 1991:6:1279-83.

++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Experiment I Wishart, G J.; Method for assessing fowl sperm quality - tent number: 6794125 Filing date: 9 Jul 2002; Issue date: 21 Sep 2004; Inventor: Graham John Hardman Wishart; Assignee: University of Abertay Dundee,; Primary Examiner: Leon B. Lankford, Jr. Attorney: Drinker Biddle & Reath LLP; Application number: 10/110,132 U.S. Classification 435/2; 435/4; 435/18; 435/29 International Classification : A01N 102) MOTILITY MOTILITY MOTILITY + + LIVE ACROSOME LIVE ACROSOME INTACT SPERM INTACT SPERM + HIGH MITOCHONDRIAL FUNCTION In the following two examples, the ability of semen to reduce MTT tetrazolium to its coloured formazan was compared with other tests of sperm quality and fertilising ability. It was found 8

that MTT reduction was highly correlated with sperm ATP content (r=0.92); sperm mobility to penetrate Accudenz (r=0.79); sperm:perivitelline layer interaction (r=0.86) and fertilising ability (r=0.74). +++++++++++++++++++++++++++++++++++++++++++++++++++++

A) Acrosome

B) Mitochondria

Live bull sperm stained simultaneously with MitoTracker Green FM and Hoechst 33342. The image contributed by Peter Sutovsky, Oregon Regional Primate Resource Center, Oregon Health Sciences University, and used with the permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.2 See Organelles and Cytoskeleton for more information on MitoTracker dyes for mitochondrion-specific staining. 9

C) Multi sperm Parameters: One technique that addresses the above issues is
flow cytometry for assessment of sperm quality as it measures multiple sperm attributes from thousands (50,000) of spermatozoa in less than 1 minute (J. K. Graham 2001) without the need to wash o.

B)

Lipid organization

C)

Lipid Peroxidation

Experiment I MOTILITY MOTILITY + LIVE ACROSOME INTACT SPERM MOTILITY + LIVE ACROSOME INTACT SPERM + HIGH MITOCHONDRIAL FUNCTION

In the following two examples, the ability of semen to reduce MTT tetrazolium to its coloured formazan was compared with other tests of sperm quality and fertilising ability. It was found that MTT reduction was highly correlated with sperm ATP content (r=0.92); sperm mobility to penetrate Accudenz (r=0.79); sperm:perivitelline layer interaction (r=0.86) and fertilising ability (r=0.74). 10

IN DEPTH: REPRODUCTIONTHE USE OF FROZEN SEMEN

Assessment of Sperm Quality

James K. Graham, PhD
302 2001 / Vol. 47 / AAEP PROCEEDINGS

During spermatogenesis, spermatozoa lose many of the organelles that most somatic cells possess (such as endoplasmic reticulum, lysosomes, and most of the cytoplasm) reducing the baggage they need to carry en route to the oocyte. The primary function of a spermatozoon, fertilizing an oocyte, is an integrated set of processes, which require multiple cell attributes. Therefore, although the spermatozoon contains few organelles, it remains a complicated cell, having multiple cell compartments, membrane compositions, and subcellular structures, all of which must be functioning properly for the spermatozoon to fertilize an oocyte. To complicate matters further, and of particular interest to the practitioner, during spermatogenesis the DNA of the cell is condensed in such a manner that genes cannot be expressed, hence no proteins can be made by the spermatozoon. This means the cell cannot repair itself from any cellular damage that occurs naturally or due to human interventions (semen handling, cooling, or cryopreservation). Improper handling of spermatozoa can permanently damage spermatozoa, making them infertile. The stallion spermatozoon consists of 3 major anatomical regions, the head, the middle piece, and the principle piece or tail, all of which are surrounded by a plasma membrane (for a detailed discussion of spermatozoal anatomy and function see Amann and Graham).1 The head contains the cell nucleus consisting of the cells chromosomes tightly packaged by protamine proteins which replace histones during spermatogenesis; and the acrosome, a membrane bound bag of enzymes at the anterior portion of the head which are necessary for the sperm to penetrate the zona pellucida of the oocyte. Disruption of the acrosome prior to the sperm being already bound to the zona pellucida renders a spermatozoon incapable of penetrating the zona pellucida and subsequently fertilizing the oocyte. The middle piece of the spermatozoa contains the mitochondria of the cell and provides the means of converting sugars, glucose primarily, into ATP

11

which is utilized by the fibers of the principle piece to cause the movement of the sperm. Destruction of the mitochondria, either by physical damage (from improper cooling or freezing) or chemical damage (improper pH or osmolality) can render the spermatozoa incapable of producing the ATP necessary to fertilize an oocyte. Likewise, damage to the principle piece, will alter the ability of the spermatozoa to swim to the oocyte, penetrate the zona pellucida and fertilize the oocyte.

+++++++++++++++++++++++++++++++++++++++++++++

Example 6

In the following two examples, the ability of semen to reduce MTT tetrazolium to its coloured formazan was compared with other tests of sperm quality and fertilising ability. It was found that MTT reduction was highly correlated with sperm ATP content (r=0.92); sperm mobility to penetrate Accudenz (r=0.79); sperm:perivitelline layer interaction (r=0.86) and fertilising ability (r=0.74). Materials and Methods for Examples 6 and 7

Males were bred from commercial ISA Brown hens and were 20-28 weeks at the time of the experiments. These birds were caged individually, given a 14:10 h light:dark photoperiod and fed a commercial breeders ration ad libitum.

12

Semen was collected by abdominal massage (Lake, 1957), diluted 1:4 in 0.15 mol/L NaCl with 20 mmol/L TES (N-tris(hydroxymethyl)methyl, 2 aminoethane suphonic acid), pH 7.4, and incubated shaking at 22 C. for up to 1 h before assay of MTT reduction (Hazary and Wishart, 1999), ATP content (Wishart, 1982) and sperm:IPVL interaction (Robertson et al., 1998). Samples assayed by the Accudenz sperm mobility test were 510 8 sperm per milliliter concentration and was estimated by light scattering at 550 nm in a spectrophotometer (Proman and McLean, 1996). Example 7

Birds were a White Leghorn type. Males were 70 weeks old and the hens were 64 weeks. Samples of semen were collected as above, diluted 1:4 in NaCl/TES and the sperm concentration calculated (as above). An insemination dose of 30 million spermatozoa from each male in 50 l of NaCl/TES was inseminated into 10 hens. Eggs were collected and stored for up to 5 days before incubation. Fertility was estimated by candling between days 4 and 8 of incubation. Results for Examples 6 and 7

The relationship between sperm MTT reduction potential and sperm ATP content, ability to penetrate Accudenz, hydrolytic activity towards the IPVL and fertilising ability of spermatozoa in semen samples from individual males are shown in FIGS. 4-7 respectively. These are all highly correlated.

Thus, sperm ATP and INT-reduction potential were found to be significantly correlated with the proportion of fertilised eggs laid by hens inseminated with a low dose of 10 million spermatozoa. This low dose of spermatozoa was useful to exaggerate differences in sperm fertilising ability, since the relationship between the number of inseminated spermatozoa and the proportion of fertile eggs laid by inseminated hens is saturatingso that differences in the 13

fertilising ability of semen samples of different quality are minimised at higher insemination doses.

An alternative to proportion of fertilised eggs for measuring sperm function in vivo is to measure, in the laid egg, the numbers of intravaginally-inseminated spermatozoa which reach and penetrate the perivitelline layers of the egg at fertilisation (see Wishart, 1999). This can be shown to be linearly correlated with the insemination dose of spermatozoa under conditions in which the proportion of fertile eggs is asymptotic (Wishart, 1995; Wishart and Staines, 1999) and is therefore a useful measurement for sperm function in vivo. Thus, the numbers of spermatozoa which interact with the perivitelline layers in vivo have been shown to be linearly correlated with the sperm quality test of their ability to hydrolyse the perivitelline layer in vitro, under conditions in which the relationship of these parameters with fertilising ability was asymptotic and non-linear (Robertson et al., 1998). The high correlation of MTT-reductive ability with in vitro sperm:perivitelline interaction ( FIG. 3 ) links the former test with the above assays of sperm function in vivo.

The fertilising ability of chicken spermatozoa has been reported to be correlated with sperm motility, measured by subjective scoring systems (Cooper and Rowell, 1958; Wilson et al., 1979), and by objective light-scattering techniques (Wishart and Palmer, 1986). More recently, a simple sperm mobility assay, based on the ability of spermatozoa to penetrate, and thus increase the turbidity of, solutions of the viscous polymer, Accudenz, has been applied to test poultry sperm quality (Froman and McLean, 1996). On the basis of this assay, semen from males was divided into average and high mobility groups, which differed by 10% in the proportion of fertilised eggs laid by inseminated hens (Froman et al., 1997). Our current findings, that MTT-reduction by semen from individual males is highly correlated with their ability to penetrate Accudenz, also adds credence to the value of the MTT reduction test as a predictor of domestic fowl fertilising ability. 14

Certain of the above parameters of individual male domestic fowl sperm quality have been demonstrated to be repeatable within different ejaculates taken from the same male and to be quantitative traits which describe a normal distribution. Furthermore, since these same studies have demonstrated up to 10-fold differences in sperm quality parameters in populations of males, these tests are valuable tools for selecting or de-selecting males to optimise fertility or cut down on the numbers of males used for breeding programmes.

Modifications and improvements can be incorporated without departing from the scope of the invention. REFERENCES

Barbato, G. F., Cramer, P. G & Hammerstedt, R. H. (1998) A practical in vitro sperm-egg binding assay which detects subfertile males. Biology of Reproduction 58: 686-699.

Bilgilli, S. F., Renden, J. A. & Sexton, T. J. (1985) Fluorimetry of poultry semen: its application in the determination of viability, enzyme leakage and fertility. Poultry Science 64: 1227-1230.

Chaudhuri, D & Wishart, G. J. (1988) Predicting the fertilizing ability of avian semen: the development of an objective calorimetric method for assessing the metabolic activity of fowl spermatozoa. British Poultry Science 29: 837-845.

Chaudhuri, D., Wishart, G. J., Lake, P. E. & Ravie, O. (1988) Predicting the fertilizing ability of avian semen: a comparison of a simple calorimetric test with other methods for predicting the fertilizing ability of fowl semen. British Poultry Science 29: 847-851.

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Cooper, D. M. & Rowell, J. G. (1958) Relations between fertility, embryonic survival and some semen characteristics in chicken. Poultry Science 37: 699-707.

Froman, D. P. & McLean, D. J. (1996) Objective measurement of sperm motility based upon sperm penetration of Accudenz. Poultry Science 75:776-784.

Froman, D. P., Feltman, A. J. & McLean, D. J. (1997) Increased fecundity resulting from semen donor selection based upon in vitro sperm motility. Poultry Science 76: 73-77.

Froman, D P & Feltman, A J. (1998) Sperm mobility: a quantitative trait of the domestic fowl ( Gallus domesticus ). Biology of Reproduction 58: 379-384.

Lake, P. E. (1957) Fowl semen as collected by the massage method. Journal of Agricultural Science, Cambridge 49: 120-126.

Lake, P. E. & Stewart, J. M. (1978) Artificial insemination in poultry. Ministry of Agriculture Fisheries and Food, Bulletin 213 . HMSO , London.

McDaniel, C. D., Hannah, J. L., Parker, H. M., Smith, T. W, Shultz, C. D. & Zumwalt C. D. (1998) Use of a sperm analyzer for evaluating broiler breeder males. 1. Effects of altering sperm quality and quantity on the sperm motility index. Poultry Science 77: 888-893.

Mossman, T. (1983) Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. Journal of Immunological Methods 65: 55-63.

Scuderio, D. A., Shoemaker, R. H., Paull, K. D., Monks, A., Tierney, S., Nofziger, T.H. , Currens, M. J., Seniff, D. & Boyd, M. R. (1988) Evaluation of a soluble tetrazolium/formazan 16

assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Research 48: 4827-4833.

Robertson, L., Wilson, Y. I., Lindsay, C. & Wishart, G. J. (1998) Evaluation of semen from individual male domestic fowl by assessment of sperm:perivitelline interaction in vitro and in vivo. British Poultry Science 39: 278-281.

Wilson, H. R., N. P. Piesco, E. R. Miller & W. G. Nesbeth (1979) Prediction of the fertility potential of broiler breeder males. World's Poultry Science Journal 35: 95-118.

Wishart, G. J. (1982) Maintenance of ATP concentrations in and of fertilising ability of fowl and turkey spermatozoa in vitro. Journal of Reproduction and Fertility 66: 457-462.

Wishart, G. J. (1985) Quantitation of the fertilizing ability of fresh compared with frozen and thawed fowl spermatozoa. British Poultry Science 26: 375-380.

Wishart, G. J. (1989) Physiological changes in fowl and turkey spermatozoa during in vitro storage. British Poultry Science 30: 443-454.

Wishart, G. J. (1993) Techniques for semen quality determination. Proceedings of the 3 rd International Symposium on Turkey Reproduction , pp 83-89. Raleigh, N.C.

Wishart, G. J. (1994) Vaginal sperm transport and sperm selection. Proceeding of the 9 th European Poultry Conference , pp 61-164. Glasgow.

Wishart, G. J. & Palmer, F. H. (1986) Correlation of the fertilizing ability of semen from individual male fowl with sperm motility and ATP content. British Poultry Science 27: 97-102. 17

Robertson, L., Wilson, Y. I., Linday, C. and Wishart, G. J. (1998) Evaluation of semen from different individual male domestic fowl by assessment of sperm:perivitelline interaction in vitro and in vivo. British Poultry Science 39:278-281.

Wishart, G. J. (1995). New approaches to evaluating male and female fertility, in:

Bakst, M. R. & Wishart, G. J. (Eds) First International Symposium on the Artificial Insemination of Poultry pp.207-223. (Illinois, USA, Poultry Science Association)

Wishart, G. J. & Palmer, F. H. (1986) Correlation of the fertilizing ability of semen from individual male fowl with sperm motility and ATP content. British Poultry Science 27: 97-102. +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

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