Effect of Season and Age on Indian Red Jungle Fowl (Gallus Gallus Murghi) Semen Characteristics: a 4-Year Retrospective Study B.A

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Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study B.A. Rakha, M.S. Ansari, S. Akhter, Elisabeth Blesbois

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B.A. Rakha, M.S. Ansari, S. Akhter, Elisabeth Blesbois. Effect of season and age on Indian red jungle fowl (Gallus gallus murghi) semen characteristics: A 4-year retrospective study. Theriogenology, Elsevier, 2017, 99, pp.105-110. 10.1016/j.theriogenology.2017.05.016. hal-01603284

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HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Copyright Version postprint Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred legal disclaimersthatapplytothejournalpertain. note thatduringtheproductionprocesserrorsmaybe discoveredwhichcouldaffectthecontent,andall copyediting, typesetting,andreviewoftheresultingproof beforeitispublishedinitsfinalform.Please our customersweareprovidingthisearlyversionofthe manuscript.Themanuscriptwillundergo This isaPDFfileofanuneditedmanuscriptthathas been acceptedforpublication.Asaserviceto Theriogenology Indian redjunglefowl( Please citethisarticleas:RakhaBA,AnsariMS,AkhterS,BlesboisE,Effectofseasonandageon Accepted Date: Revised Date: Received Date: To appearin: Reference: DOI: PII: B.A. Rakha,M.S.Ansari,S.Akhter,E.Blesbois characteristics: A4-yearretrospectivestudy Effect ofseasonandageonIndianredjunglefowl( Accepted Manuscript jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 (2017),doi:10.1016/j.theriogenology.2017.05.016. 21 May2017 21 May2017 19 December2016 Theriogenology THE 14121 10.1016/j.theriogenology.2017.05.016 S0093-691X(17)30255-8 Gallus gallusmurghi Comment citer cedocument:

) semencharacteristics:A4-yearretrospectivestudy, Gallus gallusmurghi ) semen Version postprint 25 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 9 8 7 6 5 4 3 2 1 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred

Email: [email protected] Email: +92-300-2020625 Mobile: Raw Mehr Arid Shah, Agriculture Pir Ali University, Management Department of Wildlife Bushra AllahRakha * Title: Running France Nouzilly, 37380, 4 Pakistan 3 Pakistan 2 Pakistan Rawalpindi-46300, 1 B.A. Rakha Research Paper Original jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. eatet f idie aaeet Pr er l Sha Ali Mehr Pir Management, Wildlife of Department Corresponding Author: Corresponding C des et Reproduction la de Physiologie UMR85 INRA, Agric Arid Shah Ali Mehr Pir Zoology, of Department Lyal Sargodha, of University Zoology, of Department Effect of season and age on Indian red jungle fowl fowl age red jungle Indian and season on Effectof Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 1 *, M. S. Ansari S. M. *,

ACCEPTEDEffect characteristics season semen andon age of characteristics: A 4-year retrospective study Astudy retrospective characteristics:4-year Comment citer cedocument: 2 , S. Akhter S. , ACCEPTED MANUSCRIPT

3 , and , E. Blesbois MANUSCRIPT alpindi-46300, Pakistan Pakistan alpindi-46300, ulture University Rawalpindi-46300, University ulture 4 (

Gallus gallus gallus Gallus murghi omportements, F-37380 Nouzilly, Nouzilly, F-37380 omportements, lpur Campus, Faisalbad-38000, Faisalbad-38000, Campus, lpur h Arid Agriculture University University Agriculture Arid h of Indian Red Jungle Fowl IndianFowl Jungle Red of )semen 1 Version postprint 50 50 49 48 47 46 45 44 43 42 41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred

semen quality; Indian red jungle Indiansemenred quality; fowl Keywords: year atbankingthe four age seaso the spring in of ejaculates semen that suggested is It sub-species. great that production sperm of evolution an exhibit It year. the over all noticed was production sperm integri acrosomal and parameter viability integrity, sperm membrane for season best the was Spring age. of exception the with 0.05) < (P age of years four obse was factor) quality semen integrity, acrosomal ejacula per number sperm total concentration, sperm quantitativ sperm most of age with trend increasing facto quality semen and (%) integrity acrosome (%), (1x10ejaculate per number conc (µL), volume viz; parameters semen qualitative to 6 from fowls jungle red Indian adult eight from murghi gallus characteristics semen on winter) and autumn summer, t evaluate to designed was study 4-year A programs. spe at-risk of potential production maximum achieve have to pre-requisite is it Thus, species. same the bu species avianrelated closely in only not differ ex is males adult the of potential reproductive The ABSTRACT jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 ee caatrsis Ae Sao; uniaie se Quantitative Season; Age; characteristics; Semen ACCEPTED (n=1148 ejaculates Semen manner. retrospective in ) Comment citer cedocument: 9 mL ACCEPTED MANUSCRIPT -1 ), motility (%), viability (%), plasma membrane in membrane plasma (%), viability (%), motility ),

t even in subspecies, breeds and/or strains of strains and/or breeds subspecies, in even t n. n. sperm motility that was not affected by the by affected not was that motility sperm pected to vary with age/season and largely and age/season with vary to pected rved. The highest values were observed at at observed were values highest The rved. ly differs in many points from other fowl fowl other from points many in differs ly MANUSCRIPT is concluded that Indian red jungle fowl jungle red Indian that concluded is te, plasma membrane integrity, viability, viability, integrity, membrane plasma te, of highest quality achieved for semen semen for achieved quality highest of ad ulttv tat (ee volume, (semen traits qualitative and e knowledge of seminal parameters to to parameters seminal of knowledge ty (P < 0.05), however a remarkable remarkable a however 0.05), < (P ty is for cies he effect of age and season (spring, (spring, season and age of effect he 4 ots f g. uniaie and Quantitative age. of months 54 nrto (1x10 entration f nin e jnl fw ( fowl jungle red Indian of r were recorded. A chronological chronological A recorded. were r vz vlm, oiiy plasma motility, volume, viz; s x iu n vitro in situ ex ) were regularly collected collected regularly were ) men quality; Qualitative Qualitative quality; men 9 mL -1 ), total sperm sperm total ), conservation Gallus Gallus tegrity tegrity 2 Version postprint 75 75 74 73 72 71 70 69 68 67 66 65 64 63 62 61 60 59 58 57 56 55 54 53 52 51 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred

may affect fertilizing capacities accordingly [26-2 accordingly capacities fertilizing affect may wit varies quality semen Nevertheless, [8,9,23-25]. in used either cocks the of potential reproductive integrity, acrosome and membrane plasma viability, ejacul semen the of characteristics The [17,21,22]. si without time of period infinite for state frozen situ ma to biotechnology reproductive assisted important Cryo-banking [14-20]. ameliorated and/or mitigated natural their in decline continuous facing are that th of one considered is conservation,banking semen appropriate adopting fowl domestic [7,13]. with genetichybridization de habitat are reduction population in contributing unex sudden to due decline population and diversity fragment in areas restricted in found is population Pakis Kashmir, and Jammu Azad and India Northern to jun red Indian subspecies, fowl five From [6]. wild Sp andAnimal Threatened Plant of Singapore list in Spec Threatened of native habitat [2,5]. its continuouslyin declining List Red IUCN in species concern Th [1-3]. fowl domestic the Phasianida of family of member a fowl, ancestors jungle red The the of one 1. jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study.

osrain rga [,] I i wl established well is It [8,9]. program conservation Introduction Introduction Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 The decline in Indian red jungle fowl needs special needs fowl jungle red Indian in decline The ACCEPTED Comment citer cedocument: x situ ex ACCEPTED MANUSCRIPT osrain taeis 21,2. For [2,11,12]. strategies conservation

endangered as considered fowl jungle Red is 8]. The success of semen banking depends depends banking semen of success The 8]. s, and is more likely to collapse of genetic genetic of collapse to likely more is and s, h age and season of collection in birds that birds in collection of season andage h

habitat and the threats cannot be easily easily be cannot threats the and habitat MANUSCRIPT gle fowl ( fowl gle e most applicable options for bird species species bird for options applicable emost ecies, facing high risk of extinction in the in ofextinction risk ecies, high facing ate viz; volume, concentration, motility, motility, concentration, volume, viz; ate gnificant decline in fertilizing potential potential fertilizing in decline gnificant of valuable male germplasm could be an be could germplasm male valuable of struction, egg collection, predation and and predation collection, egg struction, nan eei dvriy and diversity genetic intain aua o atfca bedn program breeding artificial or natural e red jungle fowl is enlisted as least least as enlisted is fowl jungle red e pected threats [2,11-13]. The factors factors The [2,11-13]. threats pected tan [7-10]. The Indian red jungle fowl fowl jungle Indianred The [7-10]. tan semen quality factor determine the the determine factor quality semen that avian semen can be stored in in stored be can semen avian that attention; several reports suggest suggest reports several attention; e and genus and e Gallus gallus murghi gallus Gallus e [] u is ouain is population its but [4] ies Gallus Gallus ex situ in vitro vitro in situ ex is considered considered is in vitro ex ex vitro in ) is native native is ) 3 Version postprint 100 100 99 99 98 97 96 95 94 93 92 91 90 89 88 87 86 85 84 83 82 81 80 79 78 77 76 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred

Pksa Mtooia Dprmn, 0221) The 2012-2015). Department, Metrological (Pakistan of temperature average The period. study the during a mm/summer 224.73 mm/spring, 39.2 mm/autumn, 73.13 millimeters. 1,249 about is region this of rainfall (-2.8 winter and 46.5°C) to (21.5°C summer 23.7°C), autumn seasons; four having region subtropical and moun by traversed valleys rich with tracks mountain th of slopes Southern in located is East) 0" 4' 73° a reared were fowl jungle red Indian of chicks The Th Rawalpindi. University Agriculture Arid Shah Ali Experimentalbirds 2.1 2 plasma membrane acrosomequalityandindex, integri numb sperm total concentration, sperm volume, semen winter) and summer spring, (autumn, season and age production potential of for at-risk species maximum is parameters seminal retrospective about knowledge subspec five of characteristics semen in variations Sca [30]. species same the of strains and/or breeds rel closely only in not differ largely seasons; and perfo reproductive The [17,20,29]. cryopreservation the of quality initial the on depend that outcomes froze acceptable and cryopreservation successful on jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study.

Materials and methods Materialsand Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 was study retrospective 4-year present, Therefore, ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

ated bird species but also even in subspecies, in even also but species bird ated

e North-Western Himalayas including large large including Himalayas North-Western e The average rainfall in each season was was season each in rainfall average The rce available literature depicted immense immense depicted literature available rce

MANUSCRIPT ex situ in vitro exin situ rmance of the male fowl varies with age age with varies fowl male the of rmance the area ranges from -3.9°C to 48.3°C to -3.9°C from ranges area the ejaculate to be used in processing for for processing in used be to ejaculate ty) of Indian red jungle fowl. Indianredty)of jungle e Rawalpindi region (33° 36' 0" North, 0" 36' (33° region Rawalpindi e (13.9°C to 33.5°C), spring (10.1°C to to (10.1°C spring 33.5°C), to (13.9°C tain rivers. This region falls in humid humid in falls region This rivers. tain n thawed semen quality and fertility fertility and quality semen thawed n e o rd uge ol 83] The [8,31]. fowl jungle red of ies hrfr te eqiie o achieve to perquisite the therefore hcs ee et n niiul pens individual in kept were chicks °C to 20.0°C). The average annual annual average The 20.0°C). to °C on semen characteristics (motility, (motility, characteristics semen on t Avian Research Center, Pir Mehr Mehr Pir Center, Research Avian t semen viability, ejaculate, per er designed to elucidate the effect of of effect the elucidate to designed d 73 mwne recorded mm/winter 37.3 nd conservation. conservation. 4 Version postprint 101 101 124 124 123 122 121 120 119 118 117 116 115 114 113 112 111 110 109 108 107 106 105 104 103 102 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred

pr cnetain a maue b tkn 1L of 1µL mL taking 99 by in measured formaldehyde was 37% concentration Sperm of (1mL solution citrate Spermconcentration 2.2.2 Olympus Japa BX20, (x400, microscope phasecontrast on sample semen of drop a putting by 100% to 0 from subjec phosphat was sperm of motile of percentage µL The mOsm/kg). 500 in samples as semen determined was of ejaculate each of µL motility Sperm 10 mixing 2.2.1 was microlitres using volume in measured micropipet 4- the during studied were ejaculates 1148 of total individ from collected was Semen training. of weeks succ train The collection semen 32,33]. to subjected [8, were birds Quinn The and Burrows by described evaluation quantitative and Semencollection 2.2. cockand libido. quality al on performed was cocks eight of collection Semen furt for semen sufficient get to able were we until c fecal without semen clean and neat donate to able for trained were and massage abdominal to subjected t throughout daylong the all birds the to available br cock poultry available commercially offered were t during changes significant any day-light without conditions natural to exposed cm, 121.92 x cm 106.68 jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016

Sperm progressive motility Spermprogressive motility ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

MANUSCRIPTcontinued wastraining This ontamination. e xeietl eid Te id were birds The period. experimental he he entire 4-year study period. The birds birds The period. study 4-year entire he e poesn ad xeietl work. experimental and processing her eeder feed (100g/day). Fresh water was was water Fresh (100g/day). feed eeder te. er td pro (0221) Semen (2012-2015). period study year ual birds in a graduated plastic tube. A tube. plastic graduated a in birds ual a pre-warmed glass slide (37 slide glass pre-warmed a semen collection until the birds were birds the until collection semen ternate days, depending on individual individual on depending days, ternate n). essful ejaculate was collected after 4 4 after collected was ejaculate essful f .% wv sdu ctae with citrate) sodium (w/v) 2.9% of iey vlae o a cl ranging scale a on evaluated tively ing through abdominal massage as as massage abdominal through ing and standardized environmental environmental standardized and ecie b Zmai [4 by [34] Zemjanis by described e buffer saline (pH 7.2, 300 300 7.2, (pH saline buffer e semen and 200 µL of formal formal of µL 200 and semen o C) under under C) 5 Version postprint 125 125 148 148 147 146 145 144 143 142 141 140 139 138 137 136 135 134 133 132 131 130 129 128 127 126 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred

2.2.6 stained a while the pinkish morphologicallynormal” straight and Unstained pink. and white of contrast b clear a provides mixture The immersion). oil with und slide per studied were spermatozoa 200 of total Jun Red Indian o made was smear A semen. of drop 1 of with mixed were sperm) total / Live (% Viability gluta lake the to eosin-nigrosin adding by examined 2.2.5 immersion). oil with plas un fields separate four at counted intact were spermatozoa having spermatozoa normal as classified hea swollen showing spermatozoa The glutaraldehyde. warm pre a on placed was solution incubated of drop (100mOsm/kg) and solution HOS of µL 500 with mixed mL 100 to citrate sodium of g 1 adding by prepared asses was spermatozoa of Santiago-Moreno integrity by described membrane as Plasma (HOS) test swelling membrane Plasma integrity 2.2.4 m by obtained was ejaculate per number sperm Total obtained the during theof concentration ejaculates ejaculate sperm number per Total 2.2.3. Olympusstudy throughout Japan) BX20, pe the 4-year under Germany) (Marienfeld, haemocytometer Neubauer jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016

Acrosomal integrity Acrosomalintegrity Spermviability ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

MANUSCRIPT 35]. [8, study period mate solution [37]. Twelve drops of stain of drops Twelve [37]. solution mate s “dead ” sperm. “dead ”s sperm. ed (37ed der a phase-contrast microscope (1000x (1000x microscope phase-contrast a der ackground in the smear to enhance the the enhance to smear the in ackground er a phase-contrast microscope (1000x (1000x microscope phase-contrast a er of distilled water. Semen (25 µL) was was µL) (25 Semen water. distilled of riod [8]. [8]. riod pr wr cniee a “ie and “live as considered were sperm n a glass slide, fixed and air dried. A dried. air and fixed slide, glass a n t l [6. h HS ouin was solution HOS The [36]. al. et incubated at 37at incubated s soln n cie tis were tails coiled and swollen ds, ma membrane. A total of 200 200 of total A membrane. ma hs cnrs mcocp (x400, microscope contrast phase o C) slide and fixed in buffered 2% 2% buffered in fixed and slide C) ultiplying the total volume with with volume total the ultiplying gle Fowl spermatozoa was was spermatozoa Fowl gle sed by using hypo-osmotic hypo-osmotic using by sed o C for 30 minutes. A minutes. 30 for C 6 Version postprint 149 149 172 172 171 170 169 168 167 166 165 164 163 162 161 160 159 158 157 156 155 154 153 152 151 150 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred 2.3 Statistical analysis Statistical 2.3

Meanand ± P<0.05, as presented SEM. w Differences seasons. and ages different at groups de to test difference significant least though done h Post replications. unequal with design randomized analyzed were data groups, age and seasons between a groups age different for calculated were mean) of software M statistical with Theanalyzed data were sta The excel. York,descriptive NewNew for Media, [24]. wasformula calculated the by following ejaculate semen each of (SQF) factor quality Sperm fertil of capable potentially spermatozoa of number 2.2.7 immersion. oil with magnificationof1000x microsc phase-contrast a under fields separate four 20 of total A unstained. remained acrosome ruptured uneven were spermatozoa abnormal stained; evenly be hrs. 1.5 for stain Giemsa in kept were slides Fixed for neutral in fixed and dried slide, glass clean a prepar was Smear water. mL 35 mL) into (2 atpH 7.0 sper Fowl Jungle Red Indian of integrity Acrosomal adding The by was stain prepared Giemsa[38]. stain jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Q = SQF Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016

Sperm quality factor factor Spermquality pr ocnrto Sperm concentration ACCEPTED Comment citer cedocument:

ACCEPTED MANUSCRIPT

x ejaculate volume 100

min. 30 for formaldehyde) (5% mal-saline Sperm with normal acrosome appeared to to appeared acrosome normal with Sperm

ere considered statistically different when when different statistically considered ere MANUSCRIPT ization in ejaculate. Sperm quality factor quality Sperm ejaculate. in ization Giemsa (3g) and phosphate buffer saline phosphate buffer saline and (3g) Giemsa oc comparisons between the means were means the between comparisons oc 0 spermatozoa were observed at least in in least at observed were spermatozoa 0 using analysis of variance in completely completely in variance of analysis using tistical parameters (mean, standard error error parameters standard (mean, tistical ope (x400; Olympus BX20, Japan) at at Japan) BX20, Olympus (x400; ope ed by taking a drop of semen sample on on sample semen of drop a taking by ed termine differences among individual individual among differences termine nd seasons. To compare differences differences compare To seasons. nd ly stained while spermatozoa having having spermatozoa while stained ly egastat mL matozoa was assessed through through assessed was matozoa was calculated to check the the check to calculated was ® x normal livespermatozoa version 7.25 Mc-Graw-Hill 7.25 version 7

Version postprint 173 173 197 197 196 195 194 193 192 191 190 189 188 187 186 185 184 183 182 181 180 179 178 177 176 175 174 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred

birds, their age, their environment including clima including environment their age, their birds, subsp or species furthermore, species; the of cycle semen quality high of production The [25,8,23,24]. abou clue indirect an provides (SQF) factor quality viabi integrity, membrane plasma motility, sperm of se and AI of success the programs, conservation In Semen [17]. ejaculate of quality semen initial the 4. the with spring autumn, equivalentlyin efficient and summer parameters quality the of most on recorded (P positive highly A age. of months 6-18 and 19-30 43-5 during recorded were parameters aforementioned increas the with factor quality semen and integrity me plasma in a observed was in increase an Nevertheless, increase with (P>0.05) differ not did motility v parameters quality semen qualitative on data The semen and integrity acorosme viability, integrity, ofage. winter, by followed season spring in recorded were sperm volume, ejaculate semen (P<0.05) highest The by followed age of months 43-54 ejac at higher (P<0.05) per number sperm total and concentration sperm concentration sperm volume, ejaculate on data The of months 54 to 6 from fowl jungle red of ejaculate Results 3. jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study.

Discussion Discussion Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

Sperm 2. Table in given were factor quality evaluation, based on on based evaluation, te changes such as seasons [30, 36,39-46]. [30, seasons as such changes te

MANUSCRIPT volume, Semen 1. Table in given are age ing age. The highest (P<0.05) values for for values (P<0.05) highest The age. ing autumn and summer from 6 to 54 months 54 to 6 from summer and autumn and only altered in winter. in altered and only ecies specific variations depend on the the on depend variations specific ecies ejaculates depends on the reproductive reproductive the on depends ejaculates 31-42, 19-30 and 6-18 months of age. age. of months 6-18 and 19-30 31-42, concentration and total sperm number number sperm total and concentration t the fertility potential of the males males the of potential fertility the t mbrane integrity, viability, acrosome acrosome viability, integrity, mbrane 4 months of age compared to 31-42, 31-42, to compared age of months 4 lity, acrosomal integrity and semen semen and integrity acrosomal lity, e rm t 5 mnh o age. of months 54 to 6 from ge <0.05) effect of spring season was was season spring of effect <0.05) xeto o mtlt ta was that motility of exception ulate was recorded significantly significantly recorded was ulate men cryopreservation depends on on depends cryopreservation men iz; motility, plasma membrane membrane plasma motility, iz; and total sperm number per per number sperm total and in vitro vitro in measurement measurement 8 Version postprint 198 198 222 222 221 220 219 218 217 216 215 214 213 212 211 210 209 208 207 206 205 204 203 202 201 200 199 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred

livability, acrosomelivability,attheir optim were integrity) paramet quality the of most as years 4 at maximum a wi production sperm of increase the to addition In j red Indian by produced sperm the of functionality andthrough increasing daily o low production sperm Would [47,49]. fowls domestic and [31] fowl jungle Indian in lower still were (billion) ejaculate per vol mean low to due However, conditions. management th of adaptation increasing of suggestion including hyp Many fowl. wild this in age the with production sugges to leads This old. years 4 were fowls jungle was and age, the with increased ejaculate per sperm As fowl. jungle red the ancestors, presumed the of andyearagethen decr of first the semen in volume find our to Contrary [30]. Leghorns in age the with volu Semen volume. and concentration sperm of terms be to appeared fowl jungle red Indian with results between concentration sperm chicken indigenous Thai So and [48] Chotesangasa as area tropical of breeds Nevertheless, practice. breeding the in fact known concentrat sperm in m observations decrease the from differ a greatly results reported Our [47] al. et the ofseason. best stays age and spring jungl red Indian of semen of quality and production fir the for showed we fowl, domestic to contrast In jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

um at 43-54 months of age. at 43-54 um months red jungle fowl compared to Malaysian red red Malaysian to compared fowl jungle red st time in the present study that the highest highest the that study present the in time st e species to its environment, nutrition, and nutrition, environment, its to species e eased with age, a difference with here, one one here, with a age, difference with eased a way of consequence, the total number of of number total the consequence, of way a ungle fowl increased with the age to reach reach to age the with increased fowl ungle local in modulated be would situation this

MANUSCRIPTin best the is age of years 4 as “extreme” e fowl is not reached until the fourth year year fourth the until reached not is fowl e o fo 1 o . yas f g, well- a age, of years 1.5 to 1 from ion f the longevity of sperm production? sperm of fthe longevity there be a sort of “compensation” of this this of “compensation” of sort a be there t an increase in the efficiency of sperm sperm of efficiency the in increase an t otheses may arise from this observation this from arise may otheses nseeda et al. [45] found an increase in in increase an found [45] al. et nseeda observed highest when the Indian red red Indian the when highest observed ings, broiler males showed maximum maximum showed males broiler ings, me has already been reported to differ differ to reported been already has me er measured here (Hypo-osmotic test, test, (Hypo-osmotic here measured er 1 and 2 years of age. However, our our However, age. of years 2 and 1 ume of semen, total sperm number number sperm total semen, of ume ade in most domestic fowl as Long Long as fowl domestic most in ade th the age, it was evident that the that evident was it age, the th 9 Version postprint 223 223 247 247 246 245 244 243 242 241 240 239 238 237 236 235 234 233 232 231 230 229 228 227 226 225 224 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred oet n dvlpd hg tlrne o environmen to tolerance high a developed and forest f jungle red Indian the earlier, discussed As ages. higher significantly was SQF the study, present the morphol of number parameter,the qualitative one on th on dependent is SQF The fowls. jungle red Indian be could results explain to hypothesis One studies. since limited is results our and results literature par these for However, [55]. fertility in decrease relate been has decrease Such [54,55]. roosters and age of years 1.5 and 1 between period reproductive report been previously have integrity morphological repor previous in described results the to opposite th at highest were integrity acrosome and viability sub-cellu the of integrity the succe the and for viability significance Sperm absolute of are acrosome the yearsp limiting fowl, thus or 3 4 examined old an neck naked in semen of quality the to contribute de and age; of months 6 to compared maximum was age h the to spermatozoa of response that reported [44] fertilizing and viability the maintaining in roles biochem and physical of movement the to due is tail soluti hypo-osmotic towards spermatozoa of response the with “plasticity” its increases membrane plasma Th spermatozoa age. fowl of jungle years red Indian 4 precisely, at More 37°C at solution hypo-osmotic

jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

ability of sperm [50-52]. Shanunagum et al. al. et Shanunagum [50-52]. sperm of ability owl is a resident of Temperate and Tropical Tropical and Temperate of resident a is owl

sperm and viability in decreases Indeed, ts. otential comparison with our study. ourcomparison otential study. with ameters also, the comparison between the the between comparison the also, ameters le ae wr nt xlrd n earlier in explored not were ages older at 43-54 months of age compared to other other comparedto age of months 43-54 at d dwarf chicken. But none of these studies studies these of none But dwarfchicken. d

MANUSCRIPT the Indeed, functionality. its thus and age ypo-osmotic environment at one year of of year one at environment ypo-osmotic ogically normal and live spermatozoa. In spermatozoa. live and ogically normal due to late optimal sexual maturity in in maturity sexual optimal late to due ss of fertilization. In the present study, study, present the In fertilization. of ss in free-range poultry [44], turkeys [53] [53] turkeys [44], poultry free-range in t cags n ii cmoiin and composition lipid in changes to d ae f 35 mnh, fc ta is that fact a months, 43-54 of age e e quantitative parameters of semen and and semen of parameters quantitative e ese changes would indicate that sperm sperm that indicate would changes ese on in terms of swollen head and coiled coiled and head swollen of terms in on ical compounds which play essential essential play which compounds ical d o onie ih h ed f a of end the with coincide to ed a vrain ta cud e “cost- be could that variations tal scribed that age and genetic factors factors genetic and age that scribed lar fraction of sperm such as the the as such sperm of fraction lar showed greater resistance to the the to resistance greater showed 10 Version postprint 248 248 272 272 271 270 269 268 267 266 265 264 263 262 261 260 259 258 257 256 255 254 253 252 251 250 249 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred Our study shows that the seasonal sperm production sperm seasonal the that shows study bu Our “model”, chicken indigenous Thai the from far so climates tropical Thai asin humid chicken in held consistent show breeds Some manner. specific breed environm These [30,56]. summer the of precipitation ex the by affected severely be may production sperm with climates Saharan in and desert subtropical In autumn/winte during months some for stopped totally photoperiods/temperatu of range large with climates tempe and photoperiod as such factors Environmental s and year the throughout considerably vary may and vari even and species-specific highly is production seme low remarkably a with seasons of concentration other and volume production, sperm the The in low ac and plasmatic membranes maturemuch earlier than str flagellar and pathways signaling the time; same could of functions different the where process maturation This measurements. viability the of evolution study present the in However, semen. of quality and motilit Sperm months. 30 over of chicken indigenous obt results the confirming partially study, present [31]. mat fully get would fowl jungle red Indian the that consequent and metabolism animal the for intensive”

jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 pr mtlt ws h ol qaiy atr ht was that factor quality only the was motility Sperm ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

[45,57,58]. [45,57,58]. the sperm do not reach the maturity at the the at maturity the reach not do sperm the

es among different strains or breeds [46], [46], breeds or strains different among es MANUSCRIPT ure at later ages compared to other fowls other to compared ages later at ure ie b Sned e a. 4] n Thai on [45] al. et Sonseeda by ained a smaller range of annual photoperiods, photoperiods, annual of range smaller a ental cues have also been expressed in a a in expressed been also have cues ental res, domestic fowl sperm production is is production sperm fowl domestic res, t also shows characteristics close to the to close characteristics shows also t uctures related to motility being fully fully being motility to related uctures rature affect semen production. In mild mild In production. semen affect rature rosome attributes. attributes. rosome r and reaches maximum during spring. spring. during maximum reaches and r y is a predictive parameter of viability viability of parameter predictive a is y semen production throughout the year the throughout production semen ly the sexual maturation. We suggest suggest We maturation. sexual the ly rml ht eprtrs n lc of lack and temperatures hot tremely easons among avian species [30,40]. [30,40]. species avian among easons ti prmtr i nt olw the follow not did parameter this , indicate a specific reproductive reproductive specific a indicate n volume in summer. The sperm sperm The summer. in volume n of Indian red jungle fowl is not is fowl jungle red Indian of sperm were higher in spring and and spring in higher were sperm o afce b te g i the in age the by affected not 11 Version postprint 273 273 297 297 296 295 294 293 292 291 290 289 288 287 286 285 284 283 282 281 280 279 278 277 276 275 274 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred study [68]. The reason of these differences could p could differences these of reason The [68]. study Nig of quality semen the climate,” “Saharan the In winter in sea andmaximum reported normal sperm was of [64]. photoperiod short time spermin contribut also could [67] composition phospholipids p mitochondrial in differences Seasonal [65]. semen shortened in integrity mitochondrial and energy of motilities Poor [53]. turkey and [64] poultry range with associated been winterdirectly or have autumn po opposite, the At [30,62,63]. others and domestic Leghorns in SQF and motility as well as sperm live t increase to reported been previously has increase se and spermatogenesis in role important play which the stimulates cue, environmental an as acts season t climates, se mild In best integrity). the acrosome viability, as identified man was for spring and study, traits our sperm viability In and quantitative [60]. even stressful the to mitigate cannot related that evaporation be tentatively could summer hot in semen pr sperm in reduction temporary caused temperatures domest local Nigerian and ducks pintail Northern on stu present the of results The [59]. described been hig of effects deleterious Indeed, summer. Pakistan potentially variations summer with model” “Saharan

were also reported in correlation with loss loss with correlation in reported also were he number of morphologically normal and and normal morphologically of number he dy are in agreement with previous studies studies previous with agreement in are dy h temperatures on germ cells have already already have cells germ on temperatures h he lengthening of photoperiod in spring spring in photoperiod of lengthening he photoperiod compared to spring collected collected spring to compared photoperiod MANUSCRIPT the shortening of the photoperiod in free- in the shortening the photoperiod of ossibly be attributed to heterogeneity of of heterogeneity to attributed be ossibly testosterone, and LH FSH, of secretion s f et irpin n umr season summer in disruption heat of ts oduction [40,43,59]. The low volume of of volume low The [40,43,59]. oduction y other sperm quality traits (motility, (motility, traits quality sperm other y or livability and viability of sperm in in sperm of viability and livability or related to the high temperatures of the the of temperatures high the to related e to explain the greater sensitivity of of sensitivity greater the explain to e son, which is in contrast to the present the present to contrast in whichis son, ermeability [66] and its membrane membrane its and [66] ermeability ic chicken in which high summer summer high which in chicken ic e pouto [1. Photoperiod [61]. production men fowls such as free range poultry, poultry, range free as such fowls the absence of cutaneous heat heat cutaneous of absence the erian shika brown in terms of live live of terms in brown shika erian ason for the SQF that mixes mixes that SQF the for ason 12 Version postprint 298 298 321 321 320 319 318 317 316 315 314 313 312 311 310 309 308 307 306 305 304 303 302 301 300 299 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred 4 IC (05 IC Rd it f hetnd species threatened of list Red http://www.iucnredlist.org/details/22679199/0. IUCN (2015) IUCN [4] B, Bedhom U, Gunnarsson G, Larson J, Eriksson at 2016 http://animaldiversity.org/accounts/Gallus [3] Di Animal (On-line), gallus" "Gallus Z. Gautier [2] of the world. Count DelacourThe pheasants J. [1] References Indianred fowl. jungle geneticof diversity the build to order in important very are elements These th at achieved be can quality maximal of ejaculates acrosome and membrane plasma factor, quality semen n sperm total concentration, sperm volume, of terms exhib fowls jungle red Indian that concluded is It maxi with trend increasing chronological seasonand year. present over all the s “mixed” but classical more a presents fowl jungle “Pak the parameter, this Regarding winter. to moved sea best the season, reproductive the of adaptation wo summer hot and dry terrible The climate. Saharan species and strains breeds, among quality semen the

jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. 2008;4: e1000010. 2008;4: th of origin hybrid a reveals gene skin yellow the Jense E, Randi A, Vereijken A, Jungerius D, Wright Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

MANUSCRIPT e age of four years in the spring season. season. spring the in years four of age e in months 43-54 of age quality mal atthe son of reproduction being consequently consequently being reproduction of son easonal effect since sperm production is is production sperm since effect easonal -specificity [47], but also to the specific the to also but [47], -specificity an AI program for the conservation of conservation the for program AI an umber per ejaculate, motility, viability, viability, motility, ejaculate, per umber ry Life Ltd, London1951. London1951.Ltd,ryLife istan” environment of the Indian red red Indian the of environment istan” versity Web 2002. Accessed May 31, 31, May Accessed 2002. Web versity integrity. It is suggested that semen semen that suggested is It integrity. _gallus/ _gallus/ uld have a significant role in the the in role significant a have uld Tixier-Boichard M, Strömstedt L, Strömstedt M, Tixier-Boichard it better semen quality in spring spring in quality semen better it n P, Andersson LIdentification of LIdentification Andersson P, n dmsi cikn Po Gen PloS chicken. domestic e 2015-4. 13 Version postprint 322 322 345 345 344 343 342 341 340 339 338 337 336 335 334 333 332 331 330 329 328 327 326 325 324 323 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred microsatellite markers. Anim Biotechmarkers.2015;26:201- Anim microsatellite fowl jungle red Indian between analysis structure Geneti D. Sharma J, Mathew SK, Shukla V, Kumar doi:10.4172/2157-7145.1000195. [13] forensics. wildlife in (FINS)andits implication forensicall through galliformes of Identification Goya N, Mohan LK, Sharma M, Shukla SKS, Mukesh Int1998;8:387-394. Bird Cons gallus?. [12] endangerment Genetic IL. Brisbin AT, Peterson [11] jungle( fowl red Bles I, Hussain S, Akhter MS, Ansari BA, Rakha Biol2015b;8:109-112. Res [10] (Fowl Jungle Red of quality the on frequencies Ansari S, Akhter MF, Malik I, Hussain BA, Rakha 2015a;61:379-386. [9] (Fowl Jungle Red Indian the of characteristics Akhte MF, Malik I, Hussain MS, Ansari BA, Rakha J & A pioneer Kashmir, Pak Pakistan: study. Jammu [8] ( fowl jungle red of pattern Pop NI. Dar U, Ali M, Anwar MS, Awan A, Subhani 2007;15:1–179. [7] b of checklist annotated An CJ. Hails LK, Wang http://dx.doi.org/10.2305/IUCN.UK.2012-1.RLT 2016: [6] Th gallus. Gallus (2016) International Birdlife [5]

) 2016a;174:45-55. Reprod semen. Sci Anim MANUSCRIPT e IUCN Red List of Threatened Species Species Threatened of List Red IUCN e Gallus gallus murghi gallus Gallus ) in Deva Vatala National Park, Azad Park, National Vatala Deva in ) Gallus gallus murghi gallus Gallus y informative nucleotide sequencing sequencing nucleotide informative y irds of Singapore. Raffles BullZool Raffles Singapore. of irds bois E. Cryopreservation of Indian of Cryopreservation E. bois of wild Red Jungle fowl Gallus Gallus fowl Jungle Red wild of 210. 210. Frni Rs 2013;4:195 Res Forensic J Zool 2010;42:701-706. S.T22679199A40112723.en. S.T22679199A40112723.en. ulation status and distribution distribution and status ulation and domestic chicken using using chicken domestic and dvriy n population and diversity c MS. Impact of ejaculate ejaculate of Impact MS. r S, Blesbois E. Semen Semen E. Blesbois S, r l SP, Sathyakumar S. S. Sathyakumar SP, l ). Eur J Wildl Res Wildl J Eur ). ) semen. Avian Avian semen. ) 14 Version postprint 346 346 370 370 369 368 367 366 365 364 363 362 361 360 359 358 357 356 355 354 353 352 351 350 349 348 347 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred 1983;66:2635-2644. 1983;66:2635-2644. semen to relation in quality Semen RG. Saacke [23] BiolR semen. Avian avian Blesbois E. Freezing [22] Basic WV. of of Holt frozen se aspects storage Urial( [21] ext citrate sodium and milk skim acid, Tris–citric Andrab N, Ullah S, Akhter I, Hussain BA, Rakha turkey cryosurvival.crane Ani andsperm sandhill [20] of Donoghue effects spermatozoa: DE, avian of Wildt cryopreservation G, Gee JA, Long JM, o Blanco 2011;123:242-248. Sci [19] benefits sperm crane spermatozoa: turkey on and osmoprotectantsand ATP Donoghue DE, avian Wildt cryopreservationof G, Gee JA, Long cryopre JM, semen Blanco of Theriogenology2008;69:252-261. success [18] of Predictors MM. Richard Mignon-G F, Seigneurin I, Grasseau E, Blesbois Po cryobank. French chicken:avian of creation the [17] man situ ex for cryopreservation Semen M. Boichard Limouzin I, Grasseau F, Seigneurin E, Blesbois [16] Greylag( the A, Chełmo Jerysz M, E, Chrzanowska Łukaszewicz 2000;63:1164-1171. spermatozoa.Biol Reprod [15] poul in tolerance rate cooling and cryoprotectant, Speci AM. Donoghue DE, Wildt G, Gee JM, Blanco [14]

jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 Ovis vignei punjabiensis Ovisvignei

ACCEPTED anser L.) Anser Comment citer cedocument: ACCEPTED MANUSCRIPT gander semen. Anim Reprod Sci 2004;80:163-173. 2004;80:163-173. Reprod gander Sci Anim semen.

) spermatozoa. Reprod Biol 2013;13:238-242. Biol 2013;13:238-242. )spermatozoa. Reprod MANUSCRIPT men. Anim Reprod Sci 2000;62:3-22. 2000;62:3-22. Reprod Sci Anim men. es2011;4:52-58. es2011;4:52-58. C, Besnard J, Gourichon D, Tixier- D, Gourichon J, Besnard C, m Reprod Sci 2012;131:1-8. 2012;131:1-8. Sci Reprod m ata S Sit am M Mialon- M, Jalme Saint S, rasteau enders for liquid storage of Punjab of storage liquid for enders ul Sci 2007;86:555-564. 2007;86:555-564. Sci ul i SMH, Ansari MS. Evaluation of of Evaluation MS. Ansari SMH, i ń try, eagle, and peregrine falcon falcon peregrine and eagle, try, k .Atmt n freezing skaB. on Attempts freezing and thawing rates on on rates thawing and freezing agement of genetic diversity in in diversity genetic of agement cryosurvival. Anim Reprod Reprod Anim cryosurvival. preservation. J Dairy Sci Sci Dairy J preservation. es variation in osmotic, osmotic, in variation es servation in chickens. chickens. in servation f non-permeating non-permeating f AM. Comparative Comparative AM. M Comparative AM 15 Version postprint 386 386 371 371 394 394 393 392 391 390 389 388 387 385 384 383 382 381 380 379 378 377 376 375 374 373 372 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred Poul Sci1935;14:251-253. Poul sp obtaining of method A JP. Quinn WH, Burrows Sci2013;37:564-568. Vet Anim Malaysia.Turk J [32] domestic fowl, jungle red the of quality ejaculate M, Bukar M, Nesa R, Yusoff AW, Haron A, Malik 2012;11:47-49. Sci Poult [31] leghorn white season of characteristics semen on H Mohamed SA, Makawi NA, Musharaf HAA, Elagib 2012;12:271-276. ( [30] buffalo of fertility vivo in extender acid tris-citric Glutathione-supplemented Iqb N, Ullah SMH, Andrabi BA, Rakha MS, Ansari st 2008;5 J Sci NigerianNigeria. environmentin Poul laying commercial [29] a of performance on variation OM. Alabi TE, Lawal FA, Aderemi AO, Oguntunji Publishers2001. [28] f use its and animals of semen J.The Anderson Sci1989;68:1688-1694. breederPoul cages. males in [27] testosterone, plasma fertility, semen production, RJ. Kempainen DN, Marple JA, Renden KJ, Sexton large in pa collectioninsemination and artificial [26] Neum M, Wink H, Müller M,Reinschmidt M, Lierz Res Poul Appl four morphologybreeds. chicken of J [25] Siudzi [24]

jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 ń ACCEPTED and extenders semen of Effect E. Łukaszewicz A, ska Comment citer cedocument:

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uau bubalis Bubalus MANUSCRIPT rrots ( rrots or artificial insemination. Greenworld Greenworld insemination. artificial or and carcass composition of broiler- of composition carcass and bl semtza Rpo Biol Reprod spermatozoa. bull ) cocks under Sudan conditions. Int J J Int conditions. Sudan under cocks improves the post-thaw quality and quality post-thaw the improves ermatozoa from the domestic fowl. domestic the from ermatozoa :75-82. :75-82. chicken, and bantam chicken in in chicken bantam and chicken, ann D. A novel method for semen semen formethod novel A D. ann 2008;17:101-108. 2008;17:101-108.

Psittaciformes Effects of dietary energy on on energy dietary of Effects Kasim A. Evaluation of the the of Evaluation A. Kasim l , ot V Ahe S. Akhter WV, Holt R, al rain in a derived savanna savanna derived a in rain . h efcs f g and age of effects The E. The influence of seasonal seasonal of influence The storage time on sperm sperm on time storage ). Sci Rep2013;3. Rep2013;3. ).Sci 16 Version postprint 395 395 419 419 418 417 416 415 414 413 412 411 410 409 408 407 406 405 404 403 402 401 400 399 398 397 396 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred 2006;5:838-845. 2006;5:838-845. broiler of index quality sperm the on type diluent effect CD.The McDaniel HM, Parker PR, Dumpala the Sahel Vét region in Revue of Nigeria. ÉlevMéd [41] chara Semen JD. Al-Amin SUR, Chaudhari GS, Bah genetic seasonal and purity. changes of influence [40] Gañán red-legg of Characterization E. Blesbois N, A, Toledano-Díaz C, Castaño J, Santiago-Moreno Nat Sci2006;11:447-4 J Uni cryopreservation.Wuhan [39] of effects and Methods Z. Yiling L, Jianzhong s 1997;11-19. HC Cecil MR, determinationEd.Bakst evaluation, [38] semen for Techniques curve. standard con sperm of Determination HC. Cecil MR, Bakst 2009;88:2661-2669. Sci rangePoul poultry. [37] sperm seasonal of evaluation the stainingtoimprove hypo-osmot the of Use JL. Campo López-SebastiánA, Gómez MA, Coloma C, Castaño J, Santiago-Moreno ReprodNutDévelop1981;21:1113-1124. [36] ra cockerels in ejaculates first the output after Brillard M, Garreau J, Williams M, Reviers De and Company1970.Wilkins Williams [35] therapeutic and techniq ZemjanisDiagnostic R. jungle 2016b;9:207-2012. Res spermatozoa.Avian Biol red [34] Indian of storage liquid for extenders Akhte M, Anwar I, Hussain MS, Ansari BA, Rakha [33]

MANUSCRIPT ue in animal reproduction. animal reproduction. in ue Baltimore, sd ne dfeet photoschedules. different under ised Poul Sci2015;94:80-87. Poul JP. Predicting the adult dailysperm adult the Predicting JP. ed partridge ( partridge ed Trop 2001;54:153-158. Trop2001;54:153-158. seo C LpzSbsin A, López-Sebastián MC, Esteso of semen storage temperature and and temperature storage semen of variation in native Spanish free- Spanish native in variation breeder semen. Int J Poult Sci Sci Poult J Int semen. breeder 50. 50. cteristics of local breeder cocks cocks breeder local of cteristics S Bebi ECmaio of E.Comparison Blesbois S, r ic swelling test and aniline blueandaniline test swelling ic Hongshan cock spermatozoa spermatozoa cock Hongshan ol ( fowl Bue A Tldn-íz A, Toledano-Díaz A, -Brunet centration II. Establishing a a Establishing II. centration emen storage, and fertility fertility and storage, emen als als murghi gallus Gallus Alectoris rufa Alectoris ) sperm: sperm: ) 17 ) Version postprint 420 420 442 442 441 440 439 438 437 436 435 434 433 432 431 430 429 428 427 426 425 424 423 422 421 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred temperatures on fertility of temperaturesfertility frozen on turkey spermat degree various of Effects EF. Graham PM, Zavos [50] Anim Farm in Reproduction ESE. Hafez B, Hafez chicken fertility on NatSci Thai native flock. in [49] cock ratio, mating of Effects R. Chotesangasa Sci2010;89:966-973. function.Poul sperm [48] pedigree of effect cryopreservation: semen Rooster S S, Saxena J, Pelaez DC, Bongalhardo JA, Long 1117-1122. 2014;7: World [47] of attributes sperm post-washing and relationship qua Semen D. Sharma PE, Praveena RR, Churchil Study. Vet Med2013;43:347. TheThai J Indige [46] Thai in Characteristics Semen on Breeds and Effect B. Laopaiboon T, Vongpralub P, Sonseeda 2012;52:964-968. [45] climat tropical under chicken dwarf and neck naked Eff SR. Rao MR, Reddy U, Rajkumar M, Shanmugam 2010;9:195-198. [44] breed broiler indigenous Iranian in semen quality Correl T. Mohammadabadi M, Chaji S, Tabatabaei cock breederShikabrown characteristics seminal of [43] Dze JO, Ayo PI, Rekwot BI, Onyeanusi JA, Obidi [42]

jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Williams and Wilkins, USA2000. and Wilkins, Williams Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

MANUSCRIPT 2001;35:122-131. 2001;35:122-131. number in the flock and breeder age age breeder and flock the in number ozoa. Cryobiol1983;20:553-559. Cryobiol1983;20:553-559. ozoa. ettar P, O’Sullivan NP, Fulton JE. JE. Fulton NP, O’Sullivan P, ettar s. IntSci2008;7:1219-1223. Poul s. J s of Environmental Factors, Ages Ages Factors, Environmental of s als. 7th ed. New York. Lippincott York. New ed. 7th als. s of supercooling and nucleation nucleation and supercooling of s ation between age of rooster and and rooster of age between ation line and male age on postthaw postthaw on age male and line Vet roosters. Red Island Rhode r hces J nm e Adv Vet Anim J Chickens. er nda T. Seasonal variations in in variations Seasonal T. nda c odtos Ai Po Sci Prod Anim conditions. ic ect of age on semen quality in in quality semen on age of ect nous Chickens: A One-year One-year A Chickens: nous iy aaees ter inter- their parameters, lity 18 Version postprint 443 443 467 467 466 465 464 463 462 461 460 459 458 457 456 455 454 453 452 451 450 449 448 447 446 445 444 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred Genotypes on Thermotolerance in Thai Native Chick Native Thai in Genotypes on Thermotolerance Aeagwa S, Katavatin M, Duangjinda C, Duangduen ( [59] qu semen and testosterone LH, of patterns Seasonal Ware W, Lynch TL, Herzog DE, Wildt LM, Penfold 2007;6:427-430. [58] cros chickens indigenous thai chickens, indigenous te environmental high of Effects W. Aengwanich 1999;55:47-61. [57] desc a sow: the of fertility on effects management Tuovinen M, Heinonen RJ, Love OAT, Peltoniemi Reprod1997;57:976-980. [56] fertili and composition lipid spermatozoan between Piz BK, Speake RC, Noble KA, Kelso S, Cerolini wee 1996;106:201- 60 206. to [55] 25 from fowl broiler of semen in changes Spe NC, Sparks RC, Noble S, Cerolini KA, Kelso 2003;59:753-764. [54] sperma stored and fresh of quality and composition E. Blesbois M, Magistrini D, Hermier V, Douard P and Methods membraneSpermatogenesis: integrity. [53] swellin hypo-osmotic The RS. Jeyendran S, Ramu [52] spermatozo of Response TST. Seleem AH, Daader [51]

jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Sharm El-Sheikh, Egypt, 2005;177-181. 2005;177-181. Egypt, El-Sheikh, Sharm 4 The Proc: In test. swelling osmotic hypo Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 Anas acuta Anas ACCEPTED ). Reprod Fertil Develop2001;12:229-235. Fertil ).Reprod Comment citer cedocument: ACCEPTED MANUSCRIPT

MANUSCRIPT Clim., Hot in Prod. Rabbit on Con. Inter. mperature on blood indices of Thai of indices blood on mperature Reproductive period affects lipid lipid affects period Reproductive zi F, Cavalchini LG. Relationship Relationship LG. Cavalchini F, zi a of different breeds of rabbits to to rabbits of breeds different of a sbred and broilers. Int J Poul Sci Poul J Int broilers. and sbred ty during aging of chickens. Biol chickens. of aging during ty tozoa in turkeys. Theriogenologyturkeys. in tozoa ality in the Northern pintail duck pintail Northern the in ality itv suy Ai Rpo Sci Reprod Anim study. riptive L, Derrickson SE, Monfort SL. Monfort SE, Derrickson L, ake BK. Lipid and antioxidant antioxidant and Lipid BK. ake sperm of evaluation for test g rotocols, 2013;21-25. 2013;21-25. rotocols, en and Broilers. In Proceedings Proceedings In Broilers. and en V, Saloniemi H. Seasonal and and Seasonal H. Saloniemi V, s f g. Rpo Fertil Reprod J age. of ks nich W. Effect of hsp70 hsp70 of Effect W. nich 19 Version postprint 468 468 492 492 491 490 489 488 487 486 485 484 483 482 481 480 479 478 477 476 475 474 473 472 471 470 469 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred Conference. 2005;1-10. Conference.2005;1-10. Ve Nigerian In: cocks. breeder Red Island Rhode of J VO, Sekoni AA, Anyam YU, Abubakar PI, Rekwot IntegrPhy Mol A: Part Biochemistryand Physiology [68] ( trout rainbow of muscle red from mitochondria day of Effect H. Guderley E, Kraffe N, Martin Mo Biochemist andB:Part Physiology Biochemistry depressi [67] metabolic during mitochondria liver frog Bioener EA. Belyaeva LV, Emelyanova MV, Savina Sci2006;85:232-236. [66] ar what cryopreservation: semen Avian JA. Long 2012;47:578-583. Anim Semen.Reprod Domest [65] Fr the on Season of Influence JL. Campo MT, Prieto Santiago P held eighteen temperatureturkey on semen hours. [64] semen poultry Beltsville TJ. Sexton AF, Giesen 1978;57:277-284. [63] Chicken of Capacity Fertilizing the on Components Exte Semen Poultry New A TA Fewlass TJ, Sexton Sci2000;79:1650-1668. control.Poul [62] commerc in Spermiogenesis N. Korn RJ, Thurston ;20:139-156. 1976 [61] an in meteorology of signifiance The W. Bianca Domesti 2008;241-245. in [60] Thermotolerance on Symposium 4th the of

jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 ‐ ACCEPTED Toledano C, Castaño J, Moreno Comment citer cedocument: ACCEPTED MANUSCRIPT

‐ MANUSCRIPTLópez MA, Coloma A, Díaz Oncorhynchus mykiss Oncorhynchus te ilgcl hlegs. Poul challenges?. biological the e length on oxidative capacities of of capacities oxidative on length oul Sci1983;62:1305-1311. oul extender. 9. Effect of storage storage of Effect 9. extender. and anatomy species: poultry ial siol2009;152:599-603. siol2009;152:599-603. eezability of Freeof eezability getic parameters of lamprey and and lamprey of parameters getic ml rdcin It Biomet J Int production. imal Semen Stored at 5° C. Poul Sci Poul C. 5° at Stored Semen l Biol2006;145:296-305. Biol2006;145:296-305. l atau J, Magaji S. Spermiogram S. Magaji J, atau on and activity. Comparative Comparative activity. and on nder 2. Effect of the Diluent Diluent the of Effect 2. nder terinary Medical Association Association Medical terinary c Animals. Khon Kaen. Kaen. Khon Animals. c ‐ ). Comparative Comparative ). Range Poultry Poultry Range ‐ Sebastián A, Sebastián 20 Version postprint 12 12 11 10 9 9 8 7 6 5 4 3 2 1 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred ejaculate(1x10 spermTotal per (1x10 Sperm concentration

y with x, w,differentand row = z) (age superscrip ±The for(meangiven values parameter a semen SE) Semen volume (µL) junglefowl:A 4-year retrospective study(n=8cocks Table1: seasonEffectof andage ±SE)(Mean qua on

jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 9 mL -1 ) 9 ACCEPTED ) Comment citer cedocument: umr 0.86 ± 0.2 Summer itr 0.53 ± 0.1 Winter 41.1 ± 4.1 Winter umr 25.3 ± 4.3 Summer Winter Summer Spring Spring 1.11 ± 0.2 Spring 88.7 ± 9.5 Spring uun 0.45 ± 0.1 Autumn uun 41.9 ± 5.1 Autumn Season Autumn Autumn ACCEPTED MANUSCRIPT 0.02b,y 0.02b,y 0.02b,x 0.12a,z 6-18 19-30 31-42 43-54 43-54 31-42 19-30 6-18 Age in months 0.03b,y 0.03b,y

b,z a,z a,z b,z b,y d,z a,z c,z /1148ejaculates) 0.91 ± 0.91 0.1 ± 1.83 0.3 ± 78.5 7.6 1.14 ± 1.14 0.1 ± 0.95 0.2 ± 26.4 2.8 44.2 ± 44.2 2.9 ntitativeefficiency spermatogenesisof Indianin r 56.5 ± 56.5 4.4 t differ t (P<0.05). significantly

in a column (season = a, = b, anda, c, d) and in a column (season MANUSCRIPT 0.04b,y 0.14b,x 0.25b,w 0.25b,w 0.13c,w 0.37a,w 0.14b,x 0.07b,x 0.21a,x 0.04b,y 0.04b,x 0.15a,y 0.06b,y 0.14b,x 0.22b,w 0.22b,w 0.14b,x 0.06b,y b,y b,y a,y b,y c,y d,y a,y b,y 2.50 ± 2.50 0.2 ± 63.3 5.4 106.6 ± 6.8 2.37 ± 2.37 0.2 ± 2.00 0.3 ± 2.09 0.3 ± 25.6 1.9 ± 68.0 7.5 a,x a,x a,x a,x b,x c,x b,y a,x 3.01 ± 3.01 0.2 ± 2.54 0.1 ± 3.00 0.2 ± 39.9 3.6 93.8 ± 93.8 4.2 ± 73.4 2.9 3.41 ± 3.41 0.3 160.1 ± 160.1 8.2 ed 1 b,w b,w a,w b,w b,w d,w c,x a,w

Version postprint 14 14 13 17 17 16 15 Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred

y with x, w,differentand row = z) (age superscrip ±The for(meangiven values parameter a semen SE) 4-yearretrospectiveA study 1148 (n=8 cocks/ejacu Table2: seasonEffectof andage ±SE)(Mean qua on jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. integrity (%) Sperm acrosomal (%) Sperm livability Spermmotility (%) Semen factor quality integrity (%) membrane Sperm plasma Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 ACCEPTED Comment citer cedocument: itr 41.9 ± 3.7 winter 17.8 ± 2.7 Summer itr 23.9 ± 3.0 winter 26.8 ± 4.4 Summer 21.3 ± 1.8 Summer umr 61.9 ± 2.2 Summer itr 36.0 ± 1.1 Winter uun 48.7 ± 7.2 Autumn 19.9 ± 3.1 Autumn itr 17.8 ± 2.4 winter Spring Spring 52.8 ± 4.6 Spring 61.9 ± 6.0 Spring 44.0 ± 3.4 Spring 77.8 ± 2.4 Spring uun 74.8 ± 1.7 Autumn Season Summer Summer winter Autumn Autumn uun 16.7 ± 1.9 Autumn ACCEPTED MANUSCRIPT 81.8 ± 18.0 6-18 19-30 31-42 43-54 43-54 31-42 19-30 6-18 Age in months 6.2 ±6.2 1.6 6.3 ±6.3 1.2 25.4 ± 11.3

c, c, z z b,z b,z b,z a,y b,z d c,x a,y b,x b,x b,x c,z a,y a,x b,z b,y a,y 14.1 ± 14.1 2.6 ± 7.0 1.3 ,z lates) a b,z 57.1 ± 57.1 6.3 ,z 39.6 ± 39.6 7.0 ± 38.1 3.9 ± 29.5 4.1 ± 45.8 3.8 ± 27.3 3.0 ± 42.1 5.5 ± 66.8 2.2 ± 82.1 2.7 64.4 ± 64.4 4.2 ± 54.9 7.1 ± 31.3 4.6 75.7 ± 75.7 1.7 28.0 ± 28.0 4.7 litativesemen parameters Indianin red junglefowl 69.5 ± 69.5 4.3 47.3 ± 47.3 5.6 109.0 ± 109.0 26.8 22.0 ± 7.1b,y 71.6 ± 71.6 10.3 ± 22.0 7.1b,y t differt (P<0.05).significantly

in a column (season = a, = b, anda, c, d) and in a column (season MANUSCRIPT d, b,y b,y c,y a,y c,y d,y c,x a,y b,x c x x x x b,y b,y a,y y ,y 17.5 ± 17.5 2.4 ± 47.5 7.0 ± 51.0 4.5 ± 51.7 4.4 ± 51.7 6.5 44.9 ± 44.9 3.5 ± 56.9 2.8 ± 48.3 5.1 ± 71.5 4.4 ± 83.9 5.0 64.2 ± 64.2 10.1 ± 84.7 1.5 51.4 ± 51.4 2.9 ± 47.7 6.7 ± 46.9 3.4 ± 76.9 2.9 44.2 ± 44.2 5.9 ± 43.6 5.0 a, y 179.6 179.6 ± 36.0

b,x b,x b,x a,x b,x c,y b,x a,y b,x c,x x x b,x b,x a,x y y 75.1 ± 75.1 2.9 ± 73.9 2.8 ± 79.5 1.4 ± 74.9 2.3 b, b,x 95.3 ± 95.3 1.5 ± 58.8 3.1 ± 88.9 1.8 122.2 ± 122.2 16.5 ± 88.6 1.7 66.1 ± 66.1 5.6 ± 67.8 4.4 ± 90.9 0.8 ± 84.7 1.3 ± 78.0 4.8 ± 82.7 3.4 66.0 ± 66.0 4.7 ± 90.3 0.9 x a,x 168.0 ±14.2 168.0 ± 143.5 11.7 326.4 ± 326.4 26.9 : 2 w w w w b,w a,w a,w b,w d,w b,w a,w c,w c,x b,x a,x a,x

b,w

d a, c,w ,w w

Version postprint Rakha, B., Ansari,M., Akhter,S.,Blesbois, E.(2017). Effectofseason andageon Indianred jungle fowl (Gallusgallus murghi)semen characteristics: A4-year retrospective study. • • • •

Theriogenology, 99, 105-110. DOI :10.1016/j.theriogenology.2017.05.016 The reproductive potential of IJRF of highly The is variab potential reproductive of forhi season the Springproduction best was the

(IJRF) were studied over the 4-year period. the 4-year (IJRF)period. studied over were ag to respect with parameters seminal Retrospective Semen quantitative and qualitative traits increased traits andSemen qualitative quantitative ACCEPTED Comment citer cedocument: ACCEPTED MANUSCRIPT

HIGHLIGHTS HIGHLIGHTS MANUSCRIPT with the increasing age. with the increasingage. gh quality ejaculate at 4-years of age.gh ejaculateof quality4-years at le in many points from its descendants. lefrom in manyits points e/season of Indian red jungle fowl fowl jungle red Indian of e/season