Small Ruminant Research 56 (2005) 231–238

Effect of post-thawing incubation on sperm motility andacrosomal integrity of cryopreserved Garole ram semen

Anil Joshi∗, Sadhan Bag, S.M.K. Naqvi, R.C. Sharma, J.P. Mittal

Division of Physiology, Central Sheep and Wool Research Institute, Avikanagar, via Jaipur 304501, Rajasthan, India

Received 10 June 2003; received in revised form 20 January 2004; accepted 14 June 2004

Abstract

The Garole is a prolific, but less well known small Indian sheep breed found in the hot and humid Sunderban region of WestBengal. A recent finding based on DNA tests provides new evidence that the major gene for prolifacy (FecB) in Booroola Merinostrain was introduced from the Garole sheep. The breed has been shown to have the potential to perform well in a semi-aridtropical climate and could serve as a source of valuable germplasm for incorporating prolificacy traits in the monotocus breedsof semi-arid and tropical regions of India. The aim of the present study was to evaluate sperm motility characteristics andacrosomal integrity of Garole ram spermatozoa cryopreserved under controlled conditions and subjected to a 4 h post-thawingthermal evaluation test. Diluted semen samples were packaged in mini size straws and cryopreserved under controlled conditionsusing a programmable freezer. The frozen straws were thawed at 50◦C for 10 s and incubated at 37◦C for 4 h. Sperm motilitycharacteristics and acrosomal integrity were evaluated at hourly intervals following incubation. Prior to incubation, the meanm line in thep to zoa withi cant (0 seful fore .©

K

f

eepca-lit-3;a-

0

otility and spermatozoa with intact acrosomes was 77.8 and 64.7%, respectively. There was a progressive decercentage motility and percentage normal acrosomes over time. The post-thawing incubation had a significant (P< 0.05) effecn motility and percentage of spermatozoa with normal acrosomes. After 4 h of incubation, the motility and spermato

ntact acrosomes was 56.5 and 56.7%, respectively. The effect of replication on post-thawing incubation was signifiP <.5) on motility, but not on the acrosomal integrity of spermatozoa. The information generated in this study could be ux situ preservation of Garole ram spermatozoa in straws under controlled conditions for use in artificial insemination2004 Elsevier B.V. All rights reserved.

eywords:Sheep; Garole; Semen; Freezing; Post-thawing incubation; CASA analysis

∗ Corresponding author. Tel.: +91 1437 220165;ax: +91 1437 220163.

E-mail address:aniljoshi@cswri.raj.nic.in (A. Joshi).

1. Introduction

The Garole is a rare and less well-known shbreed in India, weighing 10–14 kg at maturity, butpable of producing multiple births with an averageter size of 2.27 lambs (Ghalsasi and Nimbkar, 199Fahmy and Mason, 1996). The natural habitat of G

921-4488/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.smallrumres.2004.06.013

232 A. Joshi et al. / Small Ruminant Research 56 (2005) 231–238

role sheep is the low lying, hot and humid, Sunderbanregion of West Bengal, situated within 87◦–89◦E longi-tude and 21◦–23◦N latitude, with an average rainfall of1750 mm per annum (Bose and Moitra, 1995). Garolesheep are characterized by light brownish coat colour,coarse wool and well-developed udder. Apart from thehigh prolificacy and ability to thrive well under hotand humid climatic conditions, the other unique fea-tures of this breed are, e.g. all year breeding, grazinghabits in knee-deep water, resistance to foot rot andgood mothering ability (Sharma et al., 1999). Conser-vation of sheep genetic resources in the form of sperma-tozoa, ova and embryos assume significance if a geneof major interest or effect is detected (Ponzoni, 1997).The single autosomal Booroola fecundity (FecB) genein Booroola Merino strain is known to increase ovu-lation rate and litter size (Montgomery et al., 1992).The high prolificacy in sheep carrying theFecBgeneis the result of a mutation in the BMPIB receptorthat is located in the region containing theFecB lo-cus (Wilson et al., 2001). An earlier report indicatedthat the origin of theFecBgene may be traced backto early importation of sheep in the late 18th centuryfrom Bengal (Montgomery et al., 1994). The recentfinding based on DNA tests confirms the historicalrecords about the introduction of theFecBgene in Aus-tralian Merino flocks via the Garole sheep (Davis et al.,2002).

The Garole sheep were introduced in the semi-arid tropical environment for exploring the possibilityo ds( ons l-iG un-d e ofGas ,2c di-l theb la-t re-i .5%( r-t enf m.

After semen processing and initial microscopic eval-uation, semen is often subjected to a thermal re-sistance test involving in vitro incubation for sev-eral hours followed by re-examination to estimatethe capacity of the sperm to survive in the femalereproductive tract and retain their fertilizing ability(Fiser et al., 1991). The duration of motility andother sperm characteristics during the post-thawingincubation period are indications of the usability ofthe semen (Saacke and White, 1972). The mainte-nance of a higher motility of sperm during incuba-tion reflects a greater likelihood to survive in the fe-male genital tract and undergo capacitation and fer-tilize the ovum (Fiser et al., 1991). Exposure offrozen–thawed spermatozoa to the thermal resistancetest reveals damages, which are not apparent imme-diately after thawing but are useful in assessing thefertilizing ability of ram sperm (Aisen et al., 2000).The aim of the present study was therefore, to cryop-reserve semen of Garole rams in mini straws undercontrolled conditions and subsequently evaluate themotion characteristics and acrosomal integrity of thesperm at hourly intervals during 4 h post-thawing in-cubation.

2. Materials and methods

2.1. General

andW in-t n thef farmc farmia thes l ise usta Ga-r kg( dera as se-m ctedf aze8 withs ramsw y).

f incorporating prolificacy trait in monotocus breeSharma et al., 1999). The observations recordedexual behaviour (Maurya et al., 1999) and semen qua

ty of adult rams (Joshi et al., 1999, 2003) showed thearole rams to have a potential to perform weller semi-arid environment. The ejaculate volumarole ram semen is very low (Joshi et al., 1999a)veraging 0.58 ml (Joshi et al., 2003) and it can betored successfully in the liquid (Joshi et al., 1999b001) and frozen state (Joshi et al., 2001). Artifi-ial insemination of non-prolific ewes using freshuted Garole semen has successfully resulted inirth of highly fertile crossbred lambs. The cumu

ive ewes lambing after initial insemination andnsemination of those returning to estrus was 80Naqvi et al., 2002). However, there is a need to fuher investigate the viability of frozen–thawed semor extensive utilization of this valuable germplas

The study was conducted at the Central Sheepool Research Institute at Avikanagar during the w

er season when there are no breeding activities oarm. The main season for natural breeding at theommences at the onset of the autumn season. Thes located at longitude of 75◦28′E, latitude of 26◦26′Nnd an altitude of 320 m above mean sea level inemi-arid tropical part of the country. The rainfalrratic and mainly precipitates from July to Augnd ranges from 400 to 700 mm per annum. Adultole rams (n = 10) weighing between 11.0 to 17.014.6± 2.18), 2–5 years of age and maintained unsemi-intensive management system, were useden donors. The donor rams were randomly sele

rom the original flock. The rams were allowed to gr–10 h daily on natural vegetation interspersedeasonal shrubs and forbs. In addition to grazing,ere provided with a concentrate of 150 g/(ram/da

A. Joshi et al. / Small Ruminant Research 56 (2005) 231–238 233

2.2. Semen collection, evaluation and freezing

Semen collections were made in quick successionfrom all rams using the artificial vagina. Each ramwas scheduled for two to three ejaculations on theday of collection. The study was replicated within aweek, using the same rams for semen collection. Foreach replicate the semen quality was assessed subjec-tively for volume, consistency and wave motion (0–5scale). Ejaculates with a volume of≥0.3 ml, a thickconsistency and rapid wave motion (≥4) were pooledand the rest was discarded. The pooled semen sam-ple was assessed for sperm concentration using a pre-viously calibrated colorimeter and then diluted withthe aid of test-yolk-glycerol extender (Schmehl et al.,1986) to a final concentration of 1× × 109sperm/ml(Joshi et al., 2001). The pH and osmolality (mea-sured by automatic cryoscopic osmometer) (Osmo-mat 030, Gonotec, Berlin) of the diluent was 7.20 and1553 mOsmol/kg, respectively. Diluted semen sam-ples were aspirated into 0.25 ml French mini strawsand equilibrated at 5◦C for 2 h. Equilibrated strawswere then frozen under controlled conditions in a pro-grammable cell freezer (R-204, Planer Products Ltd.,UK), precooled to 5◦C up to−125◦C at the rate of25◦C/min and then plunged into liquid nitrogen forstorage at−196◦C until required (Joshi et al., 2001;Bag et al., 2002a).

2.3. Incubation of spermatozoa

car-r vei ngsw eret h.T fromf lasst ,2

2c

e-d 2-,3 d byt

Hamilton-Thorne motility analyzer (HTM-S version7.2 Y, USA). Prior to semen analysis, each samplewas diluted to approximately 25× 106 spermatozoa/mlin a normal saline solution (Joshi et al., 2001). Theanalyzer was briefly set-up as follows: image type:phase contrast; digitization rate: 25 frames/s; digitiza-tion time: 0.8 s; minimum contrast: 8; minimum size: 6;low/high size gates: 0.6–1.8; low/high intensity gates:0.6–1.8; magnification: 2.17. Ten microlitres aliquotsof diluted semen were placed in a prewarmed 10�mMakler counting chamber (Sefi Medical Instrument,Haifa, Israel), and loaded into the analyzer at 37◦C.At least three fields were counted for each sample. Thesperm motility characteristics included in the analy-sis included the percent motility, percent rapid motilespermatozoa (fraction of all cells moving with aver-age path velocity >25�m/s), percent linearity, percentstraightness, curvilinear velocity (VCL,�m/s), aver-age path velocity (VAP,�m/s), straight line velocity(VSL, �m/s), beat cell frequency (BCF, Hz) and lateralhead displacement (ALH,�m) of spermatozoa (Joshiand Mathur, 1996). Semen samples were also evalu-ated for acrosomal integrity as described byWatson(1975). The CASA determinations and percentage nor-mal acrosomes were derived from six observations ineach replicate and post-thawing incubation time.

2.5. Statistical analysis

The values reported in percentages were subjectedt yzedb pe-r dell pro-g et r-m levelo t at<

3

ono y ofG un-d in-c of

Incubation of frozen–thawed Garole sperm wasied out in a 5× 2 factorial design experiment and fincubation periods (0, 1, 2, 3, 4 h) and two freeziere evaluated (I and II replicates). Frozen straws w

hawed individually at 50◦C for 10 s in a water bathawed semen samples of each replicate obtained

our straws were pooled and transferred into a gubes (5 ml) and incubated at 37◦C for 4 h (Bag et al.002b).

.4. Assessment of post-thawing semenharacteristics and acrosomal integrity

The motility characteristics of the sperm immiately after thawing and subsequently after 1-,- and 4-h post-thawing incubation were assesse

he CASA technique (Bag et al., 2002b), using the

o an arc sin transformation. The data was analy two-way analysis of variance for incubationiod and replication using Harvey’s mixed moeast squares and maximum likelihood computerramme (Harvey, 1990). The Duncan’s multiple rang

est, as modified byKramer (1956)was used to deteine the difference between the two means. Thef significance differences between means was seP0.05.

. Results

In Table 1, the effect of post-thawing incubatin motion characteristics and acrosomal integritarole spermatozoa cryopreserved in mini strawser controlled conditions is set out. The effect ofubation time was significant on motility, fractions

234 A. Joshi et al. / Small Ruminant Research 56 (2005) 231–238

Table 1Effect of post-thawing incubation on motion characteristics and acrosomal integrity of Garole ram spermatozoa frozen under controlled conditions

Effect %Motility %Rapid %Linearity %Straightness %Normal acrosomes

Incubation time,H (h)0 62.6a 48.7a 47.9a 57.5 54.1a1 56.2b 41.3b 45.1b 55.6 52.1b2 53.3b 40.7bd 46.6c 57.1 51.0bd3 52.6bd 39.8bd 46.6c 57.0 50.3cde4 49.3cd 37.1cd 46.9cd 57.0 49.4ceS.E.M. 1.43 1.36 0.46 0.45 0.42Significance P < 0.05 P < 0.05 P < 0.05 n.s. P < 0.05

Replication (R)I 57.2a 45.0a 46.3 56.5 51.6II 52.4b 38.0b 46.9 57.2 51.2S.E.M. 0.91 0.86 0.29 0.29 0.27Significance P < 0.05 P < 0.05 n.s. n.s. n.s.

H × R interaction0 h, I 63.7 53.0 48.0 57.4 54.50 h, II 61.4 44.4 47.7 57.5 53.81 h, I 58.1 43.5 44.9 55.4 52.21 h, II 54.2 39.1 45.3 55.9 51.92 h, I 57.3 44.8 45.3 56.2 50.82 h, II 49.3 36.6 48.0 58.1 51.63 h, I 55.1 43.5 46.1 56.6 50.63 h, II 50.2 36.1 47.1 57.5 50.14 h, I 51.5 40.5 47.3 56.8 49.74 h, II 47.0 33.7 46.5 57.1 49.1S.E.M. 2.03 1.93 0.64 0.64 0.59Significance n.s. n.s. n.s. n.s. n.s.

Values are the least square means of the arc sin transformed values of the data (%) and in the same column with different letters differ significantly.

rapid motile sperms, linearity and normal acrosomes.The mean motility of spermatozoa immediately afterthawing was 77.8% (arc sin transformed value given inTable 1is 62.6) but after 4 h of post-thawing incuba-tion 56.5% (arc sin transformed value given inTable 1is 49.3). At 0 h the spermatozoa with normal acrosomewas 64.7% (arc sin transformed value given inTable 1is54.1), which decreased to 56.7% (arc sin transformedvalue given inTable 1is 49.4) after the 4 h incuba-tion period. The effect of replication was significanton motility and fractions of rapid moving sperms, butwas not significant regarding linearity, straightness andnormal acrosomes. However, there was no significantinteraction between incubation time and replication inall the traits.

Table 2summarizes the effect of post-thawing in-cubation on sperm kinematics of the frozen–thawedGarole ram spermatozoa. Incubation time had no sig-nificant effect on VCL, VAP, VSL, ALH and BCF of the

spermatozoa. Replication had a significant (P < 0.05)effect on all the overall means derived from CASA,except BCF. In all traits there was no significant inter-action between incubation time and replication.

4. Discussion

The importance of preserving rare breeds and es-tablishing genetic resource banks of frozen semenis becoming increasingly recognized, especially forits potential in responding to environmental changes(Holt, 1997) and the extensive utilization of superiorgermplasm for A.I. (Yoshida, 2000). Sheep breeds dif-fer markedly in fecundity (Davis et al., 1998) and inbreeds of low fecundity, substantial biological and eco-nomic advantages can be gained by the introductionof a major gene for fecundity from the more prolificbreeds (Walling et al., 2002). Garole sheep is currently

A. Joshi et al. / Small Ruminant Research 56 (2005) 231–238 235

Table 2Effect of post-thawing incubation on sperm kinematics of Garole ram spermatozoa frozen under controlled conditions

Effect VCL (�m/s) VAP (�m/s) VSL (�m/s) ALH (�m) BCF (Hz)

Incubation time,H (h)0 101.1 77.7 59.9 6.1 10.01 100.4 73.6 54.8 6.6 10.62 101.3 74.9 56.0 6.6 10.33 104.9 77.5 58.5 6.5 10.04 103.7 77.8 58.3 6.5 9.9S.E.M. 3.58 2.74 2.19 0.19 0.19Significance n.s. n.s. n.s. n.s. n.s.

Replication (R)I 109.2 81.6 61.5 6.9 10.1II 95.2 71.0 53.5 6.0 10.2S.E.M. 2.26 1.73 1.38 0.12 0.12Significance P < 0.05 P < 0.05 P < 0.05 P < 0.05 n.s.

H × R interaction0 h, I 110.2 85.0 65.8 6.5 9.80 h, II 92.0 70.3 54.0 5.6 10.21 h, I 106.3 78.0 58.5 7.2 10.51 h, II 94.5 69.2 51.2 6.0 10.72 h, I 104.0 76.8 55.8 7.2 10.42 h, II 98.7 74.0 56.2 5.9 10.13 h, I 111.1 81.7 62.1 7.0 10.23 h, II 98.7 73.3 54.8 6.0 9.84 h, I 114.3 87.3 65.2 6.6 9.44 h, II 93.2 68.3 51.5 6.4 10.3S.E.M. 5.06 3.87 3.09 0.27 0.27Significance n.s. n.s. n.s. n.s n.s.

Values are least square means and those in the same column with different letters differ significantly.

India’s most valuable germplasm due to its high pro-lificacy and the ability to thrive well under harsh andadverse climatic conditions (Sharma et al., 1999). Infu-sion of the major gene for fecundity of the small Garolebreed to a relatively medium sized monotocus sheepbreed may be a chance for enhancing carpet wool andmutton production in India. The low volume of semenproduced by the Garole allows their judicious use fornatural mating of Garole ewes or A.I. of non-prolificewes using liquid semen in the main breeding season.This study indicates enormous scope for ex situ con-servation of this prolific breed in the form of cryopre-served spermatozoa by harvesting the maximum semenquantities during the non-breeding period as the recov-ery of motile spermatozoa after thawing and follow-ing a 4 h of post-thawing incubation period were morethan 70 and 50%, respectively in both the replicates.The CASA technique is accurate for objective mea-surements of sperm kinematics, if care is taken when

preparing the semen samples and proper setting of theinstrument (Davis and Siemers, 1995). Davis and Katz(1992, 1993)reported that the results of CASA to beinaccurate if the sperm count for analysis is below 20× 106 sperm/ml or above 50× 106 sperm/ml. In thisstudy, all the samples were diluted to approximately 25× 106 sperm/ml just prior to analysis and the settings ofthe instrument were kept constant for all the observa-tions so as to ensure reliability and repeatability of theresults. This ensured that (i) the time lapse between se-men dilution and the CASA analysis was short therebyenabling the spermatozoa to survive until completionof analysis and (ii) the spermatozoa concentration waswithin the recommended range for getting an accurateCASA measurement (Bag et al., 2002b).

Ram spermatozoa are sensitive to extreme changesin temperature during the freeze–thawing process. Thedegree of seminal damage depends on several fac-tors (Naqvi et al., 2001; Salamon and Maxwell, 1995,

236 A. Joshi et al. / Small Ruminant Research 56 (2005) 231–238

2000; Watson, 1995, 2000), which limit the survivalof spermatozoa during incubation (Aisen et al., 2000;Bag et al., 2002b). Under the best experimental con-ditions about half of the population of motile spermssurvive the freeze–thaw process (Watson, 1995). Thegood post-thaw recovery obtained following long-termpreservation of Garole ram spermatozoa and mainte-nance of more than 50% motile sperms after incuba-tion in both the freezing replicates in this study may beattributed to the efficacy of controlled freezing proto-col and the criteria of processing only those ejaculatesfor cryopreservation with high motility. The results ofthis study reveal the percentage of motile sperm to besignificantly reduced during incubation. Similar resultshave been reported for ram sperm frozen in mini strawsduring post-thawing incubation following the CASAanalysis (Gil et al., 2000; Bag et al., 2002b). How-ever, after 4 h of incubation more than 50% of sper-matozoa were still motile, which was similar to theresults obtained for frozen–thawed ram semen of na-tive and crossbred breeds exposed to the thermal resis-tance test (Bag et al., 2002b). The significant decline inthe percentage motile and rapidly motile sperm duringpost-thawing incubation may be due to the inability offrozen–thawed spermatozoa to generate enough ATPthrough mitochondrial respiration as a consequence ofmitochondrial aging (Cummins et al., 1994; Viswanathand Shannon, 1997) or the toxic effect of membrane-bound aromatic amino acid oxidase enzyme releasedby the from dead sperm (Shannon and Curson, 1972).

erio-r rams tfi thes ntlyd sint ato-z permv or ofm .,1e tu ato-z tudya ero-b wala ionp

The information generated from this study couldbe useful in ex situ conservation of Garole sheepgermplasm in the form of sperm stored in straws andcryopreserved under controlled conditions. The intro-duction of this prolifacy trait of the Garole sheep intomonotocus tropical breeds is possible by means of A.I.,as extreme differences between the body weight oframs and ewes does not allow for hand mating (Naqviet al., 2002). There is no study thus far on the A.I. ofewes utilizing cryopreserved Garole ram semen. Thefertility of frozen–thawed semen of Malpura breed cry-opreserved by the same freezing protocol used for theGarole semen has been tested at the institute. The lamb-ing rate achieved following laparoscopic intrauterineA.I. and transcervical A.I. were 44.4 and 38.2%, re-spectively (Naqvi et al., 2001). Further efforts are re-quired to extensively utilize the long-term preservationsemen of this prolific breed’s semen by A.I. of non-prolific sheep breeds.

5. Conclusion

The high post-thawing recovery of motile spermato-zoa and maintenance of more than 50% motility after a4 h post-thawing incubation period indicates a promisefor long-term preservation of Garole ram semen un-der controlled conditions. However, further research ef-forts are required to assess the post-thawing fertilizinga ugha b-i

A

In-s er-i Mu-n naA

R

A ffectmen

Prolonged incubation of sperm also causes detation and changes in the acrosome integrity ofpermatozoa (Pontbriand et al., 1989). The presenndings are in agreement with other researchersperm with normal acrosome declined significauring incubation and after 4 h only 56.7% (arc

ransformed value given in Table is 49.4) of spermoa had normal acrosome. The measurement of selocity has been considered as an indirect indicatitochondrial function in spermatozoa (Graham et al984) and is associated with its fertility (Budwortht al., 1988). Krzyzosiak et al. (1999)observed thander aerobic condition during incubations, spermoal velocities do not change. The results of this sre in agreement with this observation and the aic conditions were maintained for sample withdrat hourly intervals during the post-thawing incubateriod.

bility of long-term stored Garole ram semen thrortificial insemination for achieving acceptable lam

ng rate.

cknowledgements

The authors are indebted to the Director of thistitute for providing the facilities to conduct the expments. The technical assistance rendered by Mr.ir Ahmed, Mr. N.L. Gautam and Late (Ms.) Durdafridi is also acknowledged.

eferences

isen, E.G., Alvarez, H.L., Venturino, A., Grade, J.J., 2000. Eof trehalose and EDTA on cryoprotective action of ram sediluents. Theriogenology 53, 1053–1061.

A. Joshi et al. / Small Ruminant Research 56 (2005) 231–238 237

Bag, S., Joshi, A., Rawat, P.S., Mittal, J.P., 2002a. Effect of ini-tial freezing temperature on the semen characteristics of frozen-thawed ram spermatozoa in a semi-arid tropical environment.Small Rumin. Res. 43, 23–29.

Bag, S., Joshi, A., Naqvi, S.M.K., Rawat, P.S., Mittal, J.P., 2002b.Effect of freezing temperature, at which straws were plunged intoliquid nitrogen, on the post-thaw motility and acrosomal statusof ram spermatozoa. Anim. Reprod. Sci. 72, 175–183.

Bose, S., Moitra, D.N., 1995. Bengal breed of sheep in the Sunder-bans. Asian Livestock 20 (2), 16–17.

Budworth, P.R., Amman, R.P., Chapman, P.L., 1988. Relation-ships between computerized measurements of motion of frozen-thawed bull spermatozoa and fertility. J. Androl. 9, 41–53.

Cummins, J.M., Jequier, A.M., Kan, R., 1994. Molecular biologyof the human male fertility: links with ageing, mitochondrialgenetics and oxidative stress. Mol. Reprod. Dev. 37, 345–362.

Davis, G.H., Galloway, S.M., Ross, I.K., Gregan, S.C., Ward, J.,Nimbkar, B.V., Ghalsasi, P.M., Nimbkar, C., Douglas Gray, G.,Subandriyo, Inounu, I., Tiesnamurti, B., Martyniuk, E., Eythors-dottir, E., Mulsant, P., Lecerf, F., Hanrahan, J.P., Eric Bradford,G., Wilson, T., 2002. DNA tests in prolific sheep from eightcountries provide new evidence on origin of the Booroola (FecB)mutation. Biol. Reprod. 66, 1869–1874.

Davis, G.H., Morris, C.A., Dodds, K.G., 1998. Genetic studies ofprolificacy in New Zealand sheep. Anim. Sci. 67, 289–297.

Davis, R.O., Katz, D.F., 1992. Standardization and comparability ofCASA instruments. J. Androl. 13, 81–85.

Davis, R.O., Katz, D.F., 1993. Operational standards for CASA in-struments. J. Androl. 14, 385–394.

Davis, R.O., Siemers, R.J., 1995. Derivation and reliability ofkinematic measures of sperm motion. Reprod. Fertil. Dev. 7,857–869.

Fahmy, M.H., Mason, I.L., 1996. Less known and rare breed. In:Prolific Sheep. CAB International, UK.

Fiser, P.S., Hansen, C., Underhill, H., Marcus, G.J., 1991. New ther-perm.

G e ofality

G p of

G ser-zoa.

rti-I.

pp.

H ni-

H rva-

J harac-

J anal-dian

Joshi, A., Mathur, A.K., 1996. Assessment of sperm kinematics offrozen-thawed ram spermatozoa. Indian J. Anim. Reprod. 17,42–44.

Joshi, A., Bag, S., Naqvi, S.M.K., Sharma, R.C., Rawat, P.S., Mit-tal, J.P., 2001. Effect of short-term and long-term preservationon motion characteristics of Garole ram spermatozoa: a pro-lific microsheep breed of India. Asian-Aust. J. Anim. Sci. 14,1527–1533.

Joshi, A., Naqvi, S.M.K., Bag, S., Dang, A.K., Sharma, R.C., Rawat,P.S., Mittal, J.P., 2003. Sperm motion characteristics of Garolerams raised for a prolonged period in a semi-arid tropical envi-ronment. Trop. Anim. Health Prod. 35, 249–257.

Kramer, C.Y., 1956. Extension of multiple range test to groupmeans with unequal numbers of replicates. Biometrics 12, 307–310.

Krzyzosiak, J., Molan, P., Viswanath, R., 1999. Measurement ofbovine sperm velocities under true anaerobic and aerobic con-ditions. Anim. Reprod. Sci. 55, 163–173.

Maurya, V.P., Mittal, J.P., Naqvi, S.M.K., Joshi, A., 1999. Dom-inance, libido and serving capacity of Garole rams undersemi-arid region of Rajasthan. Indian J. Anim. Sci. 69, 773–775.

Montgomery, G.W., McNatty, K.P., Davis, G.H., 1992. Physiologyand molecular genetics of mutations that increase ovulation ratein sheep. Endocr. Rev. 13, 309–328.

Montgomery, G.W., Galloway, S.M., Penty, J.M., Hill, D.F., 1994.Gene mapping and reproduction in sheep. Mol. Biol. (Life Sci.Adv.) 13, 291–302.

Naqvi, S.M.K., Joshi, A., Das, G.K., Mittal, J.P., 2001. Developmentand application of ovine reproductive technologies: an Indianexperience. Small Rumin. Res. 39, 199–208.

Naqvi, S.M.K., Maurya, V.P., Joshi, A., Sharma, R.C., Mittal, J.P.,2002. Production of crossbred lambs through artificial insemi-nation of non-prolific medium size Malpura and Avikalin ewesusing fresh diluted semen of prolific micro size Garole rams.

P Piper,rna-

P ildt,eze-zoa.

S r rela-ni-–20rs,

S se-ical

S nim.

S 986.eez-6–

mal stress test to assess the viability of cryopreserved boar sCryobiology 28, 454–459.

il, J., Soderquist, L., Rodriguez-Martinez, H., 2000. Influenccentrifugation and different extenders on post-thaw sperm quof ram semen. Theriogenology 54, 93–108.

halsasi, P.M., Nimbkar, B.F., 1993. The “garole”—microsheeBengal, India. Anim. Genet. Reso. Info. 12, 73–79.

raham, E.F., Schmehl, M.K.L., Deyo, R.C.M., 1984. Cryoprevation and fertility of fish, poultry and mammalian spermatoIn: Proceedings of the 10th NAAB Technical Conference on Aficial Insemination Reproduction, 12–14 April, Milwaukee, WNational Association of Animal Breeders, Columbia, MO,4–24.

arvey, W.R., 1990. User’s guide for LSMLMW. Ohio State Uversity, Columbus, USA.

olt, W.V., 1997. Alternative strategies for the long-term presetion of spermatozoa. Reprod. Fertil. Dev. 9, 309–319.

oshi, A., Bag, S., Mittal, J.P., 1999a. Assessment of semen cteristics of Garole rams. Int. J. Anim. Sci. 14, 277–279.

oshi, A., Bag, S., Mittal, J.P., 1999b. Computer-assisted spermysis of freshly diluted and stored Garole ram liquid semen. InJ. Anim. Sci. 69, 19–20.

Asian-Aust. J. Anim. Sci. 15, 633–636.onzoni, R.W., 1997. Genetic resources and conservation. In:

L., Ruvinsky, A. (Eds.), The Genetics of Sheep. CAB Intetional, pp. 437–469.

ontbriand, D., Howard, J.G., Maximum, M.C., Stuart, L.D., WD.E., 1989. Effect of cryoprotective diluent and method of frethawing on survival and acrosomal integrity of ram spermatoCryobiology 26, 341–354.

aacke, R.G., White, J.M., 1972. Semen quality tests and theitionship to fertility. In: Proceedings of the Fourth NAAB Techcal Conference on Artificial Insemination Reproduction, 18April, Madison, WI. National Association of Animal BreedeColumbia, MO, pp. 22–27.

alamon, S., Maxwell, W.M.C., 1995. Frozen storage of rammen I. Processing, freezing, thawing and fertility after cervinsemination. Anim. Reprod. Sci. 37, 185–249.

alamon, S., Maxwell, W.M.C., 2000. Storage of ram semen. AReprod. Sci. 62, 77–111.

chmehl, M.K., Anderson, S.P., Vazques, I.A., Graham, E.F., 1The effect of dialysis of extended ram semen prior to fring on post-thaw survival and fertility. Cryobiology 23, 40414.

238 A. Joshi et al. / Small Ruminant Research 56 (2005) 231–238

Shannon, P., Curson, B., 1972. Toxic effect and action of dead spermon diluted bovine semen. J. Dairy. Sci. 55, 614–620.

Sharma, R.C., Arora, A.L., Narula, H.K., Singh, R.N., 1999. Char-acteristics of Garole sheep in India. Anim. Genet. Res. Info. 26,57–64.

Viswanath, R., Shannon, P., 1997. Do sperm cells age? A reviewof the physiological change in sperm during storage at ambienttemperature. Reprod. Fertil. Dev. 9, 321–331.

Walling, G.A., Bishop, S.C., Pong-Wong, R., Gittus, G., Russel,A.J.F., Rhind, S.M., 2002. Detection of a major gene for littersize in Thoka Cheviot sheep using Bayesian segregation analy-ses. Anim. Sci. 75, 339–347.

Watson, P.F., 1975. Use of a Giemsa stain to detect changes in acro-somes of frozen ram spermatozoa. Vet. Rec. 97, 12–15.

Watson, P.F., 1995. Recent developments and concepts in the cry-opreservation of spermatozoa and the assessment of their post-thawing function. J. Reprod. Fertil. 7, 871–891.

Watson, P.F., 2000. The causes of reduced fertility with cryopreservedsemen. Anim. Reprod. Sci. 60–61, 481–492.

Wilson, T., Xi-Yang, W., Juengel, J.L., Ross, I.K., Lumsden,J.M., Lord, E.C., Dodds, K.G., Walling, G.A., McEwan, J.C.,O’Connell, R.O., McNatty, K.P., Montgomery, G.M., 2001.Highly prolific Booroola sheep have a mutation in the intracel-luara kinase domain of bone morphogenetic protein IB receptor(ALK-6) that is expressed in both oocytes and granulosa cells.Biol. Reprod. 64, 1225–1235.

Yoshida, M., 2000. Conservation of sperms: current status and newtrends. Anim. Reprod. Sci. 60–61, 349–355.