efficacy of evaluation of rooster sperm morphology using different staining methods

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Research in Veterinary Science 85 (2008) 583–588

Efficacy of evaluation of rooster sperm morphology usingdifferent staining methods

E. Łukaszewicz *, A. Jerysz, A. Partyka, A. Siudzinska

Wrocław University of Environmental and Life Sciences, Department of Poultry Breeding, Chełmonskiego 38c, 51-630 Wrocław, Poland

Accepted 31 March 2008

Abstract

This work focused on inexpensive methods of evaluation fowl sperm morphology, based on eosin–nigrosin smears, which can deter-mine disorders in spermatogenesis and can be recommended for evaluating the fertilising potency and selecting males in flocks repro-duced by artificial insemination. Four fowl breeds (Black Minorca, Italian Partridge, Forwerk and Greenleg Partridge) were used todetermine the efficacy of sperm morphology evaluation using four eosin–nigrosin staining methods (according to Blom, Bakst and Cecil,Morisson, Jaskowski) and three examiners of different experience (high, medium, novice). There were significant (P 6 0.01) differences insperm morphology between Blom’s staining method and those of Bakst and Cecil, Morisson or Jaskowski, irrespective of fowl breed andexaminers experience. Blom stain caused sperm head swelling and showed a drastic reduction in the proportion of live spermatozoa withnormal morphology. The staining method had a greater influence on sperm morphology evaluation than the experience of the examiners.� 2008 Elsevier Ltd. All rights reserved.

Keywords: Fowl semen; Eosin–nigrosin stains; Sperm morphology

1. Introduction

There are many methods of assessing semen quality andestimating the fertilising potency of spermatozoa. Some ofthem are regarded as highly subjective, others require spe-cial laboratory facilities (Hazary and Wishart, 2001). Theevaluation of sperm motility is one of the most often usedmethod and according to Froman and Feltmann (1998),Holsberger et al. (1998) and King et al. (2000) spermatozoaneed to move within oviduct up to infundibulum – the siteof fertilization. Contrary to above, the experiments carriedout by Howarth (1983) proved that spermatozoa collectedfrom rooster testes, despite very poor motility, were able tofertilize the ovum, because the oviduct itself ensures theirmovement toward infundibulum.

0034-5288/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.rvsc.2008.03.010

* Corresponding author. Tel.: +48 71 3205 774; fax: +48 71 3205 776.E-mail address: [email protected] (E. Łukaszewicz).

For the purposes of artificial insemination, the dailysemen output, based on ejaculate volume and sperm con-centration, is commonly use to calculate the number ofinsemination doses. However, in case of ganders derivedfrom Anser anser L. species, in which the percentage of liveintact spermatozoa in freshly collected semen constitutesonly 50% of total live cells, such estimation seems to beinsufficient (Łukaszewicz, 2002; Łukaszewicz and Kru-szynski, 2003), since it says nothing about the real numberof spermatozoa capable of fertilising the ovum. Therefore,sperm morphology seems to be one of the most importantqualitative characteristics of semen (Kuster et al., 2004). Itmay be a basic indicator for predicting the fertilising abilityof spermatozoa, based upon correlation between particularcategories of spermatozoa and fertility results (Łukas-zewicz, 1988), male selection, and also semen suitabilityfor liquid storage or cryopreservation for artificialinsemination purposes (Donoghue and Wishart 2000;Łukaszewicz, 2002). Sperm morphology can also serve asan indicator of some disorders in spermatogenesis. Success

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584 E. Łukaszewicz et al. / Research in Veterinary Science 85 (2008) 583–588

in assessment of sperm morphology depends on stain prep-aration techniques, stains type and staining methods (Bil-gili et al., 1985).

Nowadays many laboratories evaluate the quality ofsemen using ‘‘objective” methods based on computer anal-ysis (CASA) or using sophisticated equipment. However,such equipment is expensive and only few research institu-tions or commercial organisations can afford to buy them,even although semen evaluation prior to its further pro-cessing or use for purposes of artificial insemination is veryimportant. Several studies (Bahr and Bakst, 1987; McDan-iel, 1995; McIntyre, 1995; Mellor, 2001) emphasize thenecessity of male selection based on semen quality. Donog-hue (1999) demonstrated that after turkey inseminationwith pooled semen collected from several toms, 18 out of26 males used as semen donors produced very few or nooffspring, resulting from specific sperm competition in fer-tilisation of ovum.

Eosin–nigrosin dye is commonly used in laboratory stainwhich allows analysis of sperm structure (Bjorndahl et al.,2003; Lake, 1978). When stained smears are viewed underthe oil immersion objective of light microscope, the percent-age of viable, live, properly formed spermatozoa, nonviableand also partially-damaged spermatozoa can be deter-mined. Live, normal spermatozoa posses an intact plasmamembrane which protects them from penetration of eosin.Under the microscope these spermatozoa appear white,unstained against the purple background of nigrosin. Deadand damaged spermatozoa have a permeable plasma mem-brane, which enables eosin penetration of the cell to staininternal organelles pink (Bakst and Cecil, 1997). Live sper-matozoa can be categorised into different forms, for exam-ple morphologically normal, or with defects in the acrosome(crooked or loose), head (bulb, small, enlarged, looped),neck (broken at different angle in relation to head), mid-piece and tail (swelling, looping, partial or totally lacking)(Blom, 1981; Edens et al., 1973; Parker et al., 1942).

There is a great variety of protocols for stain preparationand methods of making semen smears. The stain describedby Bakst and Cecil (1997) is recommended for tom andmeat-type chicken breeders. Using it they demonstratedthat good quality, fresh (unstored) semen, should havemore than 90% live and morphologically normal cells.The procedures elaborated by Blom (1981) and Jaskowski(Głod, 1976) initially were used for bull semen, however,later were also used successively for gander (Chełmonska,1972) and chicken semen (Łukaszewicz, 1988). Morissonet al. (1997) positively adopted Blom (1950) procedure forsemen evaluation of Rhode Island Red roosters using BPSEfor both semen dilution and eosin and nigrosin solution.

In semen evaluation methods, the human factor is veryimportant, with differences in results related to individualbias and experience (Bilgili et al., 1985; Root-Kustritzet al., 1998).

The aim of this study was to compare the usefulness ofdifferent eosin/nigrosin staining techniques for evaluatingthe morphology of spermatozoa from different fowl breeds

and to assess the effect of examiners’ experience on evalua-tion efficiency.

2. Materials and methods

The experiment was carried out in the Department ofPoultry Breeding of Wroclaw University of Environmentaland Life Sciences, Poland.

2.1. Birds and semen collection

Roosters of four different breeds of domestic fowl: BlackMinorca, Italian Partridge, Forwerk and Greenleg Par-tridge were used as the semen donors. Each breed was rep-resented by five sexually adult males (nine months old).The birds were kept in individual cages (60 � 50 �75 cm), at 20 �C, under 14 L/10 D photoperiod. Roosterswere provided with commercial feed for reproductivechicken and water ad libitum.

From each breed semen was collected and pooled threetimes a week by the dorso-abdominal massage method(Burrows and Quinn, 1937), 10 semen collections were per-formed for every group.

2.2. Stain and chemical composition

The examination of sperm morphology was performedon histological smears stained with eosin–nigrosin pre-pared according to:

I. Blom (1981): one part of the 5% eosin and four partsof the 10% nigrosin (dissolved in distilled water).

II. Bakst and Cecil (1997): 17.35 g sodium glutamate,1.28 g potassium citrate, 8.51 g sodium acetate,0.68 g magnesium chloride were diluted to 1 L withdistilled water and then 5 g nigrosin and 1 g eosinwere dissolved in 100 mL of this buffer.

III. Morisson et al. (1997): 1.6 g eosin and 3.0 g nigrosinwere dissolved in 100 mL BPSE diluent (BeltsvillePoultry Semen Extender) (Sexton, 1977). Composi-tion of diluent: 12.7 g potassium diphosphate �3H2O; 8.67 g sodium glutamate; 5.0 g fructose; 4.3 gsodium acetate 3H2O; 1.95 g TES [N-tris(Hydroxy-methyl)methyl-2-aminoethane sulfonic acid]; 0.64 gpotassium citrate; 0.65 g potassium monophosphate;0.34 g magnesium chloride � 6H2O were diluted to1 L with distilled water.

IV. Jaskowski (Głod, 1976): one part of 4% eosin solu-tion (4 g eosin dissolved in 96 mL of 2.9% sodium cit-rate) and three parts of 8% nigrosin solution (8 gnigrosin dissolved in 92 mL of 2,9% sodium citrate).

Chemical components were obtained from:

– Sigma-Aldrich: eosin Y, nigrosin water soluble, sodiumcitrate, potassium citrate tribasic hydrate 98%, TES P99%,

Fig. 1. Classes of spermatozoa morphology: (a) morphologically normal,(b) bulb-head, (c) looped-head, (d) protoplasmatic-droplike head, (e)double head, (f) bent-neck, (g) spermatid and (h) dead spermatozoa.

E. Łukaszewicz et al. / Research in Veterinary Science 85 (2008) 583–588 585

– Fluka: D(�)-fructose P98% (HPLC), potassium citratemonobasic 98.0%, potassium diphosphate trihydrateP99%, sodium acetate trihydrate 99.0%. sodium gluta-mate (L-glutamic acid monosodium salt monohydrate)P98%,

– POCH: magnesium chloride hexahydrate, potassiummonophosphate.

All components used for stains preparation were pureand diluted with bi-distilled water.

2.3. Techniques of preparation the histological smears

The histological smears were made within 20 min aftersemen collection. The stains were prepared exactly accordingto the methods described above, but all smears were madethe same way: 10 ll of stain was placed (with automatic pip-ette) on sterilized, defatted microscope slide, then a drop ofsemen was added. These components were gently mixedfor 30 s with glass rods and the smear was made. The slidewas left at 37.5 �C until completely dried. The stains, micro-scope slides and glass rods used in preparation were warmedto 37.5 �C on the ultrathermostat. For each preparation 300spermatozoa were evaluated (following Blom, 1981; Parkeret al., 1942). Morphology of spermatozoa was examinedunder oil immersion objective (1250� magnification) bylight microscopy (Type Jenaval, Carl Zeiss). Spermatozoawere categorised as ‘‘total live” (unstained) cells, and amongthem: morphologically normal (typical spindle-shaped headand well-marked acrosome), bulb-head, other head defor-mations (protoplasmatic-drop-like head, looped-head, dou-ble head, damaged acrosome), bent-neck, cells with otherdeformations (not included to any above types and sperma-tids – immature cells) and dead spermatozoa (all pink cellsstained by eosin) (Fig. 1). The results of morphologicalevaluation were expressed as the percentage of particularcategories of spermatozoa (300 cells = 100%).

2.4. Examiners

Semen smears were examined independently by threeindividuals of high, medium and low experience, respec-tively. Analyses were performed on the same slides, in sim-ilar visual fields. The examiners did not contact and notassist each other during evaluation.

2.5. Statistical analysis

The effect of staining method on fowl sperm morphol-ogy was presented as the average data of three examinersand four fowl breeds (n = 120).

The effect of staining technique on each particular fowlbreed was analyzed on the basis of the total data from allthree examiners (n = 30).

The effect of staining method and examiner’s experienceon sperm morphology was described on the basis of 40readings (10 for every breed).

The data obtained were analyzed using ANOVA andDuncan’s multiple range tests (SAS system, General LinearModels Procedure). All percentage data were transformedto arc sin prior to analyses.

3. Results

3.1. The effect of staining method on fowl sperm morphology

The staining methods affected sperm morphology signif-icantly. Pooling data from all breeds and examiners, themorphological picture of spermatozoa stained accordingto the Morisson method (III) showed a significantly(P 6 0.01) lower proportion of spermatozoa in the ‘‘livein total” category (91.64%). In contrast, the rest oftechniques showed higher level of this sperm type (94.01–94.93%). Staining by the Blom method (I) resulted inswelling of the sperm head, with 75.58% of spermatozoacategorised as bulb-headed, which was significantly differ-ent (P 6 0.01) from the range of 8.05–10.26% in samplesstained by other methods. Compared to Bakst and Cecil(II), Morisson (III) and Jaskowski (IV), staining methods,sperm morphology assessed according to the Blomprocedure also displayed significantly lower number of live


normal sperm and higher number of cells with bent-neckand other deformations (P 6 0.01). Within the ‘‘otherdeformations” category, cells with deformed tail were mostfrequently observed and within the category ‘‘other headdeformations” damaged acrosomes were most frequent.

3.2. Sperm morphology of four fowl breeds depending on

staining technique

Taking into consideration the morphology of spermato-zoa of the four fowl breeds, it becomes evident that themethod of semen staining technique had a significant effecton the outcome. As discussed above, in smears preparedaccording to Blom method, sperm morphology from malesof all breeds was significantly (P 6 0.01) poorer than insmears made by other techniques (Tables 1–4). In semenstained according to Blom the number of live normal cellswas extremely low (0.67% in Black Minorca – Table 1;4.98% in Italian Partridge – Table 2; 2.93% in Forwerk –Table 3 and 2.41% in Greenleg Partridge – Table 4). TheBlom procedure also resulted in a significant (P 6 0.01)increase in the number of cells with bulb-head and bent-neck in semen of all breeds evaluated. It is interesting thatfor semen of the Italian Partridge, this staining method dif-

Table 1Effect of staining method on sperm morphology of Black Minorca (n = 30; m

Staining method Classes of spermatozoa (%)

Live in total Live normal Bulb-head

Blom (I) 95.30 ± 3.96 0.67A ± 1.84 80.08A ± 6.14Bakst, Cecil (II) 95.97 ± 2.34 79.87B ± 5.88 7.61B ± 3.27Morisson (III) 94.52 ± 3.16 77.62B ± 6.78 8.48B ± 4.11Jaskowski (IV) 96.08 ± 2.62 79.02B ± 5.89 7.59B ± 3.09

Values within each column with different superscripts differ significantly (A, B

Table 2Effect of staining method on sperm morphology of Italian Partridge (n = 30;



Blom (I) 92.29A ± 4.25 4.98A ± 10.52 73.21A ± 11.42Bakst, Cecil (II) 91.06AB ± 3.96 71.92B ± 8.82 10.30B ± 8.73Morisson (III) 88.00B ± 5.88 66.93Bb ± 8.22 12.62B ± 6.66Jaskowski (IV) 92.96A ± 3.51 74.26Ba ± 6.98 9.39B ± 4.10


Table 3Effect of staining method on sperm morphology of Forwerk (n = 30; means ±



Blom (I) 94.30 ± 3.46 2.93B ± 5.57 73.53A ± 10.39Bakst, Cecil (II) 93.67 ± 2.85 75.74A ± 5.91 6.89B ± 2.95Morisson (III) 91.61 ± 4.13 71.43A ± 6.63 9.62B ± 4.24Jaskowski (IV) 94.79 ± 3.56 76.43A ± 6.44 6.84B ± 3.70


ferentiated the number of ‘‘live in total” and ‘‘live normal”cells, which was not observed for other breeds (Table 2).

3.3. Effect of staining method and examiner’s experience on

sperm morphology

Results of evaluation the fowl sperm morphology, per-formed on the basis of eosin–nigrosin histological smearsprepared by four different techniques and scored indepen-dently by three examiners revealed that the stainingmethod had a greater influence than experience of exam-iner. In relation to most spermatozoa classes there wasno significant influence of examiner experience on theamount of cells in each category. Considering the propor-tion of ‘‘live normal” spermatozoa, which are the mostimportant from a fertilising potency viewpoint, significantdifferences between examiners were observed only for theMorrison technique (P 6 0.01). This difference betweenassessment made by expert and novice examiner was7.23% points (data not presented). The less experiencedexaminers were more critical in almost all staining tech-niques and showed no difficulties in recognising ‘‘bulb-head” and ‘‘bent-neck” cells, with all staining methods.Some disagreement between examiners in relation to

eans ± SD)

Other head deformations Bent-neck Other deformations

3.37a ± 1.39 7.80A ± 4.40 3.38 ± 1.961.66b ± 0.65 1.63B ± 1.04 5.20 ± 3.131.66b ± 0.53 1.63B ± 1.52 4.92 ± 2.721.90ab ± 0.63 2.03B ± 1.61 5.53 ± 4.21

– P 6 0.01; a, b – P 6 0.05).

means ± SD)


2.88 ± 0.93 7.74A ± 2.77 3.48 ± 1.901.82 ± 0.50 2.90B ± 1.56 4.11 ± 1.941.69 ± 0.44 2.51B ± 1.59 4.24 ± 1.771.58 ± 0.35 3.19B ± 1.62 4.54 ± 1.78

– P 6 0.01; a, b – P 6 0.05).

SD)


3.52 ± 1.16 11.84A ± 5.60 2.54 ± 1.502.19 ± 0.72 5.27B ± 3.59 3.51 ± 1.927.13 ± 0.80 4.61B ± 3.32 3.56 ± 1.892.16 ± 0.62 5.43B ± 3.21 3.91 ± 2.39

– P 6 0.01).

Table 4Effect of staining method on sperm morphology of Greenleg Partridge (n = 30; means ± SD)


Live in total Live normal Bulb-head Other head deformations Bent-neck Other deformations

Blom (I) 94.16 ± 3.58 2.41B ± 6.55 75.48A ± 10.73 4.02A ± 1.30 9.72A ± 4.99 2.46 ± 1.11Bakst, Cecil (II) 95.41 ± 1.93 80.51A ± 3.43 7.57B ± 2.99 4.97B ± 0.46 2.42B ± 1.19 3.31 ± 1.32Morisson (III) 92.44 ± 4.29 75.20A ± 5.91 10.30B ± 5.27 4.77B ± 0.40 2.01B ± 0.97 3.33 ± 2.00Jaskowski (IV) 95.90 ± 2.89 79.49A ± 5.16 8.39B ± 4.09 2.14B ± 0.55 2.33B ± 1.24 3.54 ± 1.30

Values within each column with different superscripts differ significantly (A, B – P 6 0.01).

E. Łukaszewicz et al. / Research in Veterinary Science 85 (2008) 583–588 587

‘‘other head deformation” and ‘‘other deformation” cate-gories for almost all staining techniques was observedand could be attributed to examiners’ experience. Theexperience of ‘‘medium” and ‘‘novice” examiners appearedinsufficient for distinguishing the subtle differences inrespect to such deformations. However, such a relativelysmall proportion of classes of spermatozoa may beregarded as of less significance.

4. Discussion

The present experiment showed that a simple, inexpen-sive and non-invasive a method as eosin–nigrosin staining,could be used to evaluate semen quality and, as a conse-quence, to estimate the fertilising potency of spermatozoa.However, although this method is regarded as highly sub-jective, the results obtained showed that the experience ofthe examiner is less important than the choice of stainingmethod. From four tested eosin–nigrosin staining methodsit can be conclude that the Blom’s procedure should not beused for evaluation of fowl spermatozoa, since it causedsignificant sperm head swelling. In smears stained accord-ing to Blom method there was also a low percentage of live,normal spermatozoa comparing to other techniques. Bi-distilled water was used in the Blom method, whereasremaining stains were dissolved in diluents with composi-tions similar to that of seminal plasma. This observationis supported by Edens et al. (1973) who modified Blomstain (distilled water was replaced by glutamate solution)and obtained 80.5% of normal cells in fowl semen. Suchsignificant differences in sperm morphology probably werecaused by hypo-osmolarity of the medium. The osmoticpressure of three of the tested stains (Bakst and Cecil, Mor-isson and Jaskowski) was slightly higher (from 400 to520 mOsmol/kg) than the osmotic pressure of freshly col-lected semen from the four breeds, which ranged from320 to 340 mOsmol/kg. However osmolarity of the Blomstain was lower and amounted 220 mOsmol/kg. Thesehypo-osmotic conditions resulted in swelling of the spermhead. Bakst (1980) and Donoghue et al. (1996) alsoreported plasmalemmal swelling at the midpiece and tailof chicken and turkey spermatozoa, due to the uptake ofwater in similar conditions. In this work, the plasmalemmaaround the sperm head was probably more sensitive to thelower tonicity of the Blom stain resulting in more intensivewater transport inside the cell.

Of several techniques used currently for assessing semenquality, it can be stated that from the practical point ofview, one of the most critical indicators of male fertilisingpotency is the number of live, morphologically intact sper-matozoa. In this case, procedures based on evaluation ofeosin–nigrosin histological semen smear seem to be goodpredictors, even if they belong to group called ‘‘operator-dependent” methods. In the present experiment the smearswere evaluated by high experienced, medium and noviceexaminers and for three, out of four tested staining proce-dures (Blom, Bakst and Cecil, Jaskowski), there were nosignificant disagreements between examiners in relation tothe percentage of live normal cells, bulb-head and bent-neck spermatozoa, the most frequent abnormal forms infresh or stored poultry semen. Significant differenceoccurred only between the highly experienced and noviceexaminer, for smears stained according to the Morissonmethod.

Chalah and Brillard (1998) compared eosin–nigrosinstaining with SYBR14/PI (dual fluorescence) and con-cluded that the efficacy of these procedures depended onsemen treatment. In the case of fresh non-stored semen,the difference was only 3% points, while after 24 h of stor-age at 4 �C it increased to 24% points. On the other hand,the percentage of viable spermatozoa evaluated duringentire storage period by the SYBR14/PI procedure showedgreater variations than those evaluated in eosin–nigrosinsmears. For semen evaluated 24 h after freezing and thaw-ing both procedures gave comparable results. However, ithas to be pointed out that SYBR14/PI staining onlyenables observation of sperm head membrane integrity(live/dead cells) which, as it was indicated in the presentstudy, do not create difficulties in eosin–nigrosin smears,even when by a novice operator. Additionally theSYBR14/PI procedure is expensive since it requires special-ized equipment such as a fluorescent microscope. Cooperand Rowell (1958) found a high correlation between fertil-ity and the percentage of dead spermatozoa revealed byeosin–nigrosin staining. Moreover, eosin–nigrosin can beperformed in a simple farm laboratory, equipped only witha light microscope and hot plate.

Although it was not the aim of the presented experimentit is worth noting, that irrespective of staining procedure orexaminers experience, the percentage of live morphologi-cally normal cells observed in semen of all evaluated fowlbreeds was lower than in semen collected from roosters


of the commercial lines (Ansah et al., 1985; Blesbois et al.,2005 and our unpublished data), both laying and meat-type. With the exception of Greenleg Partridge breed,which is bred in Poland for eggs with lower cholesterol con-tent and kept in extensive housing systems, the remainingbreeds are kept mainly by amateurs. Lower semen quality,and low percent of fertility, can be attributed to inbreeding,since probably these breeds are reproduced without breed-ing programmes and the size of flocks managed by individ-ual ‘‘breeders” is insufficient.

In conclusions, the simple, inexpensive and not invasiveeosin–nigrosin method can be recommended for use infarms where flocks are reproduced by artificial insemina-tion with fresh semen and semen evaluation is necessarybefore female insemination.

References

Ansah, G.A., Segura, J.C., Buckland, R.B., 1985. Semen production,sperm quality, and their heritabilities as influenced by selection forfertility of frozen-thawed semen in the chicken. Poultry Science 64,1801–1803.

Bakst, M.R., 1980. Fertilizing capacity and morphology of fowl andturkey spermatozoa in hypotonic extender. Journal of Reproductionand Fertility 60, 121–127.

Blom, E., 1950. A one minute live-dead sperm stain by means of eosin–nigrosin. Journal of Fertility and Sterility 1, 176–177.

Blom, E., 1981. Physiology and pathology of reproduction. MedycynaWeterynaryjna 4, 239–242 (in Polish).

Bahr, J.M., Bakst, M.R., 1987. Poultry. In: Hafez, E.S.E. (Ed.),Reproduction in Farm Animals. Lea & Febiger, Philadelphia, pp.379–497, Chapter 18.

Bakst, M.R., Cecil, H.C., 1997. Techniques for semen evaluation, semenstorage, and fertility determination. 3. Sperm viability. I. Nigrosin/eosin stain for determining live/dead and abnormal sperm counts. ThePoultry Science Association, Inc., Savoy, Illinois, pp. 29–34.

Burrows, W.H., Quinn, J.P., 1937. The collection of spermatozoa from thedomestic fowl and turkey. Poultry Science XVI (1), 19–24.

Bilgili, S.F., Renden, J.A., Sexton, K.J., 1985. The influence of stainingtechniques and examiners on evaluation of the morphology of fowlspermatozoa. Poultry Science 64, 2358–2361.

Bjorndahl, L., Soderlund, I., Kvist, U., 2003. Evaluation of the one-stepeosin–nigrosin staining technique for human sperm vitality assessment.Human reproduction 18, 813–816.

Blesbois, E., Grasseau, I., Seigneurin, F., 2005. Membrane fluidity and theability of domestic bird spermatozoa to survive cryopreservation.Reproduction 129, 371–378.

Chełmonska, B., 1972. Seasonal changes in function of gander reproduc-tive track in aspect of artificial insemination. Part I i II. PolskieArchiwum Weterynaryjne 15, 575–611 (in Polish).

Chalah, T., Brillard, J.P., 1998. Comparison of assessment of fowl spermviability by eosin–nigrosin and dual fluorescence (Sybr-14/PI). Theri-ogenology 50, 487–493.

Cooper, D.M., Rowell, J.G., 1958. Relations between fertility, embryoticsurvival and some semen characteristics in chicken. Poultry Science 37,699–707.

Donoghue, A.M., 1999. Prospective approaches to avoid flock fertilityproblems: predictive assessment of sperm function traits in poultry.Poultry Science 78, 437–443.

Donoghue, A.M., Wishart, G.J., 2000. Storage of poultry semen. AnimalReproduction Science 62, 213–232.

Donoghue, A.M., Garner, D.I., Donoghue, D.J., Johnson, L.A., 1996.Assessment of the membrane integrity of fresh and stored turkeyspermatozoa using a combination of hypo-osmotic stress, fluorescentstaining and fowl cytometry. Theriogenology 46, 153–163.

Edens, F.W., Van Krey, H.P., Siegel, P.B., 1973. Selection for body weightat eight weeks of age 10. Spermatozoal morphology. Poultry Science52, 2287–2289.

Froman, D.P., Feltmann, A.J., 1998. Sperm mobility: a quantitative traitof the domestic fowl (Gallus domesticus). Biology of Reproduction 58,376–384.

Głod, W., 1976. Reproduction and bovine insemination, Chapter V.Semen evaluation. PWRiL, Warszawa, pp. 115–138, (in Polish).

Howarth Jr., B., 1983. Fertilizing ability of cock spermatozoa from thetestis, epididimis, and vas deferens following intramagnal insemina-tion. Biology of Reproduction 28, 586–590.

Hazary, R.C., Wishart, G.J., 2001. Assay of sperm quality in the domesticfowl by MTT reduction. British Poultry Science 42, 111–114.

Holsberger, D.R., Donoghue, A.M., Froman, D.P., Ottinger, M.A., 1998.Assessment of ejaculate quality and sperm characteristics in turkeys:sperm mobility phenotype is independent of time. Poultry Science 77,1711–1717.

King, L.M., Kirby, J.D., Froman, D.P., Sonstegard, T.S., Harry, D.E.,Darden, J.R., Marini, P.J., Walker, R.M., Rhoads, M.L., Donoghue,A.M., 2000. Efficacy of sperm mobility assessment in commercialflocks and the relationship of sperm mobility and insemination dosewith fertility in turkeys. Poultry Science 79, 1797–1802.

Kuster, C.E., Singer, R.S., Althouse, G.C., 2004. Determining sample sizefor the morphological assessment of sperm. Theriogenology 61, 691–703.

Lake, P.E., 1978. The principles and practice of semen collection andpreservation in birds. In: Symposium of Zoological Society, London,p. 43

Łukaszewicz, E., 1988. Study of diluents for cock’s semen storage in thelight of laboratory estimation and fertility rates. Zeszyty Naukowe ARwe Wrocławiu, Zootechnika XXX 168, 43–59 (in Polish).

Łukaszewicz, E., 2002. Cryopreservation of Anser anser L. gander semen.Zeszyty Naukowe AR we Wrocławiu 440, Rozprawy CXC, 1–11 (inPolish).

Łukaszewicz, E., Kruszynski, W., 2003. Evaluation of fresh and frozen-thawed gander semen of individual ganders by assessment of sperma-tozoa motility and morphology. Theriogenology 59, 1627–1640.

McDaniel, G.R., 1995. Managing broiler breeders for maximum fertility.World Poultry 9, 25–27.

McIntyre, D.R., 1995. Selling sires by the dose. In: Bakst, M.R., Wishart,G.J. (Eds.), Proceedings of First International Symposium on theArtificial Insemination of Poultry. Poultry Science Association, Savoy,Illinois, pp. 123–127.

Mellor, S., 2001. Selecting males by sperm quality. World Poultry 3, 32–34.

Morisson, M., Bordas, A., Petit, J.M., Jayat-Vignoles, C., Julien, R.,Minevielle, F., 1997. Associated effects of divergent selection forresidual feed consumption on reproduction, sperm characteristics, andmitochondria of spermatozoa. Poultry Science 76, 425–431.

Parker, J.E., McKenzie, F.F., Kempster, H.L., 1942. Fertility in the maledomestic fowl. Research Bulletin 347, 1–50.

Root-Kustritz, M.V., Olson, P.N., Johnston, S.D., Root, T.K., 1998. Theeffects of stains and investigators on assessment of morphology ofcanine spermatozoa. Journal of the American Animal HospitalAssociation 34, 348–352.

Sexton, T.J., 1977. A new poultry semen extender. 1. Effect of extension onthe fertility of chicken semen. Poultry Science 56, 1443–1446.

efficacy of evaluation of rooster sperm morphology using different staining methods

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