Effect of time and depth of insemination on fertility of Bharat Merino sheep inseminated trans-cervical with frozen-thawed semen

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1 Kumar ad Naqvi Joural of Aimal Sciece ad Techology 2014, 56:8 RESEARCH Ope Access Effect of time ad depth of isemiatio o fertility of Bharat Merio sheep isemiated tras-cervical with froze-thawed seme Davedra Kumar * ad Syed Mohammed Khursheed Naqvi Abstract Backgroud: Artificial isemiatio (AI) ca serve as a powerful tool to the sheep owers for makig rapid geetic progress of their flock. The AI i sheep is mostly performed usig fresh seme with two reasos i) lambig rate followig tras-cervical AI with froze seme is limited by the iability of froze-thawed sperm to trasit the cervix ad ii) the eed of circumvetig the cervical barrier through laparoscope aided itrauterie AI. Therefore, AI with froze-thawed seme is ot as widespread i sheep as it is i other domestic species. However, to get maximum beefits through the use of AI, froze-thawed seme is a prerequisite because istead of high fertility, the short shelf life of fresh seme coupled with a limitatio o the umber of isemiatio doses achievable per uit time restricts the widespread use of idividual sires. Therefore, i order to ehace lambig rate, a total of 240 tras-cervical artificial isemiatios with froze-thawed seme were performed i Bharat Merio ewes durig autum seaso either oce i the eveig (G-I, 10 h after oset of estrus, = 100) or twice (G-II, 14 h ad 22 h after oset of estrus, = 140) i.e. oce i the morig ad agai i the eveig. Results: The pregacy rate (proportio of pregat ewes cofirmed by ultrasoography at day 40) ad lambig rate (proportio of ewes lambed) were higher i G-II as compared to G-I (26.4 vs 20% ad 19.3 vs 10%, respectively). The differece i lambig rates was statistically (P < 0.05) sigificat. The depth of isemiatio withi cervico-uterie tract had o sigificat effect o pregacy ad lambig rates. Coclusios: The results idicate that lambig rate i sheep followig TCAI with froze-thawed seme was sigificatly iflueced by time of isemiatios. Two isemiatios after 14 ad 22 h of oset of estrus ehaced the lambig rates of Bharat Merio sheep as compare to sigle isemiatio after 10 h of oset of estrus. The TCAI techique with froze-thawed ram seme is promisig ad may serve as a valuable tool for geetic improvemet of sheep breeds. Research efforts are goig o worldwide to overcome the poor fertility followig TCAI with froze-thawed seme. Keywords: Froze seme, Ewe, No-ivasive itrauterie isemiatio, Backgroud Artificial isemiatio (AI) ca serve as a powerful tool to the sheep owers for makig rapid geetic progress of their flock. The AI is expected to play a crucial role for large-scale productio ad multiplicatio of fecudity gee carrier germplasm via itrogressio of fecudity gee ito the o-prolific sheep breeds. The AI i sheep is mostly performed usig fresh seme, sice fertility * Correspodece: davedraror@gmail.com Divisio of Aimal Physiology ad Biochemistry, Cetral Sheep ad Wool Research Istitute, Avikaagar, via Jaipur, Rajastha, Idia followig AI with froze seme is limited by the iability of froze-thawed sperm to trasit the cervix. Eve whe very large umber of motile spermatozoa is deposited i the cervix, fertility is lower for froze seme tha for fresh seme [1]. Therefore, AI with froze-thawed seme is ot as widespread i sheep as it is i other domestic species. However, to get maximum beefits through the use of AI, froze-thawed seme is a prerequisite because istead of acceptable fertility, the short shelf life of fresh seme coupled with a limitatio o the umber of isemiatio doses achievable per uit time restricts the widespread use of idividual sires Kumar ad Naqvi; licesee BioMed Cetral Ltd. This is a Ope Access article distributed uder the terms of the Creative Commos Attributio Licese ( which permits urestricted use, distributio, ad reproductio i ay medium, provided the origial work is properly credited. The Creative Commos Public Domai Dedicatio waiver ( applies to the data made available i this article, uless otherwise stated.

2 Kumar ad Naqvi Joural of Aimal Sciece ad Techology 2014, 56:8 Page 2 of 6 I all domestic species AI with froze-thawed seme gives acceptable pregacy rates oly whe seme is deposited itrauterie. I sheep, the cervix is a formidable barrier to peetratio for tras-cervical itrauterie depositio of froze-thawed seme due to its caudally facig, eccetric series of four to five fuel-like rigs [2-4]. At preset satisfactory ad reliable lambig results with froze-thawed seme are oly obtaiable by circumvetig the cervical barrier through laparoscope aided itrauterie AI [5-7]. But the cost, ivasive ature ad eed for professioal labor limits its wider use [8]. The developmet of tras-cervical artificial isemiatio (TCAI) techique has got immese potetial for usig froze-thawed seme because of its o-ivasive ature ad as a alterative techique for ivasive laparoscopeaided itrauterie AI [9]. The pregacy ad lambig rates achieved through TCAI with froze seme is ot still acceptable ad there is eed to improve lambig rates followig TCAI. Besides breed, froze-thawed seme quality, sperm umbers, ewe coditio ad time of isemiatio, the frequecy of isemiatio to meet out the availability of sperm ear ovulatio for successful fertilizatio, ad catheter peetratio (site of seme depositio) are importat factors which may ifluece success rate of TCAI. The time of isemiatio is related to the time of ovulatio. Ovulatio occurs aroud the ed of estrus. Isemiatio should be performed at a time sufficietly before ovulatio so that by the time of ovulatio a large populatio of sperm is established i the ampulla, the site of fertilizatio. I geeral, with twice daily ispectios for heat detectio, if aimal showig estrus i the morig it should be isemiated i the eveig (10 h after oset of estrus), ad if aimal showig estrus i the eveig it should be isemiated i the morig (14 h after oset of estrus). However, time of isemiatio eeds to be adjusted accordig to whether the seme is fresh or froze thawed, sice the former should have a fertilizig life i the female reproductive tract well i excess of 24 h, while for the latter the fertilizig life may ot be more tha 12 h. Therefore, with froze-thawed seme two isemiatios 8 h apart may play role i providig large populatio of live sperm (owig to the short life spa of froze-thawed sperm) at the site of fertilizatio by the time of ovulatio. Keepig this i view a fertility trial was coducted i Bharat Merio ewes to determie the ifluece of time of isemiatio (10 h Vs 14 & 22 h after oset of estrus) ad depth of isemiatio withi cervicouterie tract o pregacy ad lambig rates followig TCAI with froze-thawed ram seme. Results ad discussio The results are preseted i Table 1. The time of isemiatio had sigificat (p < 0.05) effect o lambig rates with higher values i G-II as compare to G-I. However, time of isemiatio had o sigificat effect o pregacy rates. The depth of isemiatio withi cervico-uterie tract i.e. cervical peetratio had o sigificat effect o pregacy ad lambig rates. However, both pregacy ad lambig rates were slightly higher i case of full cervical peetratio of isemiatio eedle. The success rate attaied i this study i terms of pregacy ad lambig rates followig TCAI of ewes with froze-thawed seme for oe oestrus cycle is at par with other reports [10-16]. The variatio i the results may be due to the differeces i breed, seaso, feedig, maagemet, freezig ad thawig procedures, isemiatio dose (umber of spermatozoa per straw), equipmet used for TCAI, level of peetratio of catheter ito the cervix, time ad frequecy of isemiatio i relatio to ovulatio ad oset of estrus whether atural or sychroized. Moreover, the process of maipulatig a isemiatio catheter through the cervix has bee liked to reductios i pregacy ad lambig rates. It has bee suggested that cervical trauma ad vagial/ cervical stimulatio caused by the catheterizatio may activate pathways that are associated with uterie immue defeses ad that iterrupt pregacy at its early stages [17,18,14]. It is believed that froze-thawed sperm are less motile ad lack stamia to trasverse the highly viscous cervical mucus, because of ultrastructural, biochemical ad fuctioal chages udergoe by a large sperm populatio but phagocytosis of the sperm by leukocytes is also cosidered a cause of the reduced fertility [19]. Ultrastructural chages maily affect sperm membraes because durig the froze-thawed process there is a redistributio of lipids that alters lipid-lipid ad lipidprotei relatios, which are ecessary for the ormal fuctio of sperm membraes [20]. The higher lambig rate of the ewes isemiated 14 ad 22 h after oset of estrus as compare to ewes isemiated 10 h after oset of estrus idicates that the time ad/or frequecy of isemiatio of froze-thawed seme is a critical determiat of fertility, due primarily to the sigificatly reduced lifespa of froze-thawed seme i the female reproductive tract. However, Salamo [21] reported o differece betwee the mea lambig results for frequecy of isemiatios i.e. sigle (44.4%, 59/133) ad double isemiatio (46.2%, 61/132) with froze seme. The effect of double isemiatio depeds upo the time whe the first isemiatio is performed i relatio to ovulatio. So, the effect of the double isemiatio is more reliable whe the first isemiatio is carried out at the begiig of estrus tha whe it takes place i the middle or at the ed of estrus. The beeficial effect of two isemiatio at 14 ad 22 h after oset of estrus i the preset study may be because isemiatio is artificial, atural sexual behavior is abset ad the male ad female

3 Kumar ad Naqvi Joural of Aimal Sciece ad Techology 2014, 56:8 Page 3 of 6 Table 1 Effect of timig ad depth of isemiatio withi cervico-uterie tract o pregacy ad lambig rates of Bharat Merio ewes isemiated with froze-thawed seme Timig of isemiatio after oset of estrus Depth of isemiatio withi cervico-uterie tract Full (uterie) Mid (3 rd to last cervical fold) Partial (os to 2 d cervical fold) Overall 14 & 22 h a 10 h b Overall Values with differet superscripts i same colum are sigificatly (P < 0.05) differet. reproductive evets have to be carefully sychroized to miimize the gamete waitig time before fertilizatio occurs. Moreover, two isemiatio may prevet from the possibility that isemiatio is practiced too early i relatio with ovulatio as seems i the G-I where isemiatio was doe at 10 h after oset of estrus. I the preset study the depth of cervical peetratio i.e. site of seme depositio did ot ifluece statistically (p < 0.05) pregacy ad lambig rates, however, these are slightly more i case of uterie depositio as compared with cervical depositio. Takig ito cosideratio the aatomy of the sheep cervix, a highly complex fibrous structure with may folds that obliterate the lume, it is uderstadable. I partial cervical peetratio, a part of seme or the whole quatity ca flow back ito the vagia where the acidic ph is ufavorable coditio for the sperm. I mid cervical peetratio, a large umber of sperm practically come to a dead ed i the rigs of the cervix ad caot get ito the body of the uterus. I full cervical peetratio, seme eters directly ito the body of the uterus. The depth of cervical peetratio is affected by the aatomy of cervical lume. Cervices with a less covoluted lume are more peetrable [22]. The eccetric folds are the oes most difficult to pass ad they ifluece the desig of the tip of the AI eedle [23]. Geerally, the secod fold is more misaliged tha the first ad third, which origiates a arrow cervical lume [3,24]. The depth from the os to the first 2 folds of the cervix has bee show to be the major factor limitig the cervical peetrability [25,26]. Several authors have reported that low fertility obtaied whe seme is applied via the cervix, is associated with low peetratio. The high fertility rate o itrauterie depositio of seme has also bee reported by several workers [10,12,27-30]. However, recetly it has also bee reported that the degree of catheter peetratio ito the cervix had o correlatio with fertility of ewes isemiated with froze-thawed seme [16,31]. The liear array real time ultrasoud scaig by the trasabdomial route is a reliable method for (early) pregacy diagosis i sheep with accuracy of more tha 90% [32,33]. The differece i pregacy ad lambig rates i the preset study may be due to fetal mortality or uobserved abortios that took place after day 40 of pregacy. Dixo et al. [34] reported that approximately 19.9% of the ewes experieced late embryoic loss, fetal loss, or both; ad 21.2% of the embryos or fetuses were lost from d 25 to term. The proportios of fetuses lost were associated with breed type, cocetratios of progesteroe, estradiol ad vascular edothelial growth factor. Cryoijuries of spermatozoa associated with freezig ad thawig, ad maipulatios associated with TCAI techique ca all cotribute to fetal mortality, probably due to iadequate vascular growth i the edometrium ad perhaps coceptus. Lagford et al. [35] ad Olese [36] also reported icreased embryoic mortality betwee day 18 ad term whe froze thawed seme was used. The cotributio of false positive results to the differeces betwee pregacy ad lambig rates may ot be igored. Coclusios The results of this study idicate that lambig rate i sheep followig TCAI with froze-thawed seme was sigificatly iflueced by time of isemiatios. Two isemiatios after 14 ad 22 h of oset of estrus ehaced the lambig rates of Bharat Merio sheep as compared to sigle isemiatio after 10 h of oset of estrus. The TCAI techique with froze-thawed ram seme is promisig ad may serve as a valuable tool for geetic improvemet of sheep breeds. Research efforts are goig o worldwide to overcome the poor fertility followig TCAI with froze-thawed seme. Improvemet i the freezig protocol ad the use of exogeous cervical dilatators i sheep have bee ivestigated but a much better uderstadig of sheep cervical physiology ad the mechaism of atural cervical dilatatio at estrus is required to facilitate TCAI for sheep with froze-thawed seme to achieve acceptable fertility. Methods The study was coducted with the approval of Istitute Aimal Ethics Committee (IAEC), which is i the semiarid tropical area of the Idia at E logitude, N latitude ad at a altitude of 320 m above mea

4 Kumar ad Naqvi Joural of Aimal Sciece ad Techology 2014, 56:8 Page 4 of 6 sea level. The climate of this place is typically hot with yearly miimum ad maximum temperature of 4 ad 46 C, respectively. The aual raifall rages from 400 to 700 mm with a erratic distributio throughout the year. A total of 240 TCAI with froze-thawed seme were performed i 2 4 year old Bharat Merio ewes havig body weight of kg, durig autum seaso whe major breedig activities commece at the istitute farm. Bharat Merio sheep has bee evolved at Cetral Sheep ad Wool Research Istitute, Avikaagar to cater the idigeous demad of fie wool for woolle sector by crossbreedig ative ewes (Nali, Chokla, Malpura ad Jaisalmeri) with exotic Rambouillet/Soviet Merio rams ad stabilizig the populatio at 75% exotic iheritace [37]. Twelve adult Bharat Merio rams of 3 4 years of age ad kg body weights were used as seme doors. The rams ad ewes were maitaied uder the semi-itesive maagemet system before ad after isemiatios. All the aimals were allowed for 8 10 h daily grazig o atural vegetatio iterspersed with seasoal shrubs, grasses ad forbs (Achyrathes aspera, Commelia forskalaei, Eleusie aegypticae ad Sorghum helepese). I additio to grazig, they were provided a cocetrate mixture of 300 g/day. After grazig, the aimals were housed i a chai-lik fece eclosure havig asbestos sheet roof ope from all the sides (Figure 1). The seme of idividual rams was froze well i advace ad kept cryopreserved till the day of isemiatio. O the day of freezig, ejaculates were obtaied from door rams i quick successio by artificial vagia after moutig o the restraied estrus ewe secured i the service crate. The seme samples were evaluated for volume, cosistecy, wave motio (0 5 scale), cocetratio (photometrically) ad percetage of motile spermatozoa (0-100%). Ejaculates havig thick cosistecy, rapid wave motio, 90% iitial motility ad more tha spermatozoa per ml were immediately diluted with a TESTyolk-glycerol exteder [38] at 25 C to a fial cocetratio of spermatozoa per ml. The exteded seme samples from idividual ram were froze i a Plaer R-204 programmable cell freezer uder cotrolled coolig ad freezig rates [39]. Briefly, diluted samples were aspirated ito 0.5 ml size Frech plastic straws (IMV Techologies, L Aigle, Frace), sealed with polyviyl alcohol powder, submerged i water kept i a rectagular glass tray at 25 C ad after dryig straws were loaded vertically i the programmable cell freezer pre-cooled to 25 C. Cotrolledrate coolig of straws was iitiated i the cell freezer at the liear rate of 0.15 C per mi from 25 to 5 C followed by a holdig time of 2 h at 5 C ad freezig from 5 to 125 C at the rate of 25 C per mi ad the pluged ito liquid itroge for storage util required. Thawig of idividual straws was doe at 60 C for 10 secod i a water bath just prior to TCAI. Post-thaw attributes of spermatozoa were objectively assessed by a computerassisted seme aalysis techique (Hamilto-Thor HTM-S versio 7.2 Y motility aalyzer) straw per straw. Sigle straw (0.25 ml Frech straw havig 250 millio sperm) of froze-thawed seme with more tha 50% postthaw motility was immediately used for TCAI of oe ewe. Estrus was detected i ewes twice daily (07:00 ad 19:00) by paradig adult rams, fitted with a apro. Ewes Total aimals (=240) Sigle isemiatio (After 10 h of oset of estrus) (=100) Double isemiatio (After 14 & 22 h of oset of estrus) (=140) Full (uterie) (=73) Mid (3rd to last cervical fold) (=12) Partial (osto 2d cervical fold) (=15) Full (uterie) (=89) Mid (3rd to last cervical fold) (=23) Partial (os to 2d cervical fold) (=28) Figure 1 Experimetal desig.

5 Kumar ad Naqvi Joural of Aimal Sciece ad Techology 2014, 56:8 Page 5 of 6 were isemiated for oe cycle either oce, oly i the morig (G-I, sigle isemiatio, = 100) or twice, oce i the morig ad agai i the eveig (G-II, double isemiatios, = 140). Ewes exhibitig estrus i the morig alog with ewes i estrus o the previous eveig were brought daily to the semeology laboratory for isemiatio. Ewes exhibited estrus o previous eveig were kept i G-II ad isemiated twice 8 h apart i.e. betwee 09:00 to 10:00 as well as 17:00 to 18:00 (14 h ad 22 h after oset of estrus). Ewes exhibited estrus same day i the morig were kept i G-I ad isemiated oce betwee 17:00 to 18:00 (10 h after oset of estrus). The isemiatios were made usig idividual ram seme allotted to the particular ewe as per the breedig pla (improvemet by selective breedig) of Bharat Merio sheep. The equipmet ad cradle used for TCAI were fabricated as described earlier by Naqvi et al. [12]. The ewes were restraied i the cradle i dorsal decumbecy with the hid quarter elevated so that the vulva was positioed at a agle of 80 to 90 from the groud. The isemiatio was performed throughout the study by the same isemiator usig the procedure of Naqvi et al. [12]. I brief, the speculum with pluger lubricated with small amout of medical gel was itroduced ito the vagia ad exteral os was located after removal of the pluger with the aid of the light. The eedle was getly itroduced ito cervix ad maipulated through the cervical folds ito the deepest locatio possible. A maximum time limit of 5 miutes was set to achieve full peetratio of the cervix. If full peetratio had ot bee achieved seme was deposited i the cervix. The depth of cervical peetratio was scored as: (i) os to secod fold: partial, (ii) third to last fold: mid ad (iii) eedle iserted to its full legth: full (uterie depositio). The isemiated ewes were closely observed for retur i estrus over two-cycle period ad util lambig. At 40 days of gestatio, all the isemiated ewes were examied for the presece of fetuses by usig a real time Desktop Veteriary Ultrasoud Scaer system equipped with covex array 5.0 MHz/40R/60D trasducer (Model SA- 600 V, MEDISON Compay, Limited. Korea). Data o pregacy rate (proportio of pregat ewes cofirmed by ultrasoography) ad lambig rate (proportio of ewes lambed) were recorded. The data were statistically aalyzed by chi square test as per Sedecor ad Cochra [40] to determie the effect of time of isemiatios ad depth of isemiatio withi cervico-uterie tract o pregacy ad lambig rates. Competig iterests The authors declare that they have o competig iterests. Authors cotributios DK carried out freezig, thawig ad post-thaw evaluatio of seme, made acquisitio of data, aalysis ad iterpretatio of data ad drafted the mauscript. SMKN desiged the experimet, carried out TCAI, ad give fial approval of the versio to be published. Both authors read ad approved the fial mauscript. Ackowledgemets The authors are thakful to the Director of the Istitute for providig all the research facilities ad Mr Muir Ahmed ad Mr N.L. Gouttam for rederig techical assistace durig this study. Received: 5 May 2014 Accepted: 17 July 2014 Published: 24 July 2014 Refereces 1. Maxwell WMC, Hewitt LJ: A compariso of vagial, cervical ad itrauterie isemiatio of sheep. J Agricul Sci 1986, 106: Halbert GW, Dobso H, Walto JS, Buckrell BC: The structure of the cervical caal of the ewes. Theriogeology 1990, 33: Naqvi SMK, Padey GK, Guatam KK, Joshi A, Geethalaxmi V, Mittal JP: Evaluatio of gross aatomical features of cervix of tropical sheep usig cervical silicoe moulds. Aim Reprod Sci 2005, 85: Ael L, Alvarez M, Martiez-Pastor F, Garcia-Macias V, Ael E, Paz P: Improvemet strategies i ovie artificial isemiatio. Reprod Domestic Aim 2006, 41: Salamo S, Maxwell WMC: Froze storage of ram seme. II. Causes of low fertility after cervical isemiatio ad methods of improvemet. 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