Effect of time of arti®cial insemination on fertility of
progestagen and PMSG treated indigenous
Greek ewes, during non-breeding season
A. Karagiannidis
a,*, S. Varsakeli
b, G. Karatzas
b, C. Brozos
a aClinic of Obstetrics and AI, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki,21 Kosti Palama, 551 33 Kalamaria, 540 06 Thessaloniki, Greece
bNAGREF/Institute of Reproduction and AI of Thessaloniki, 570 08 Ionia, Greece
Accepted 17 May 2000
Abstract
A total of 2567 indigenous Greek ewes (Chios, Vlachiki and VlachikiChios breeds) were used to determine the optimum
time for insemination, following synchronization of oestrus with MAP-impregnated intravaginal sponges and PMSG during non-breeding season. Within each breed group, the ewes were divided into three subgroups and submitted to a double blind cervical arti®cial insemination 48 and 60 h (subgroup I), 60 and 72 h (subgroup II) and 48 and 72 h (subgroup III) after sponge withdrawal. From the results of the present investigation it can be concluded that the conception rate in the Chios island breed is better than that in the VlachikiChios breed, with the latter being better than that in the Vlachiki breed. Using ®xed time
for AI, a better conception rate is obtained when applied 48 and 72 h after sponge withdrawal for Chios and ChiosVlachiki
breeds, while for Vlachiki breed a better conception rate is obtained when ®xed AI is applied 48 and 60 h after sponge withdrawal.#2001 Elsevier Science B.V. All rights reserved.
Keywords:Arti®cial insemination; Oestrus synchronization; Fertility; Goats
1. Introduction
Modern systems of sheep production are associated with several levels of intensi®cation, including repro-ductive management. A crucial step aiming to improvement of the reproductive performance is the intensi®cation of the rhythm of lambing, with the (3 lambings in 2 years) system being one of the most important, especially in dairy ewes. In order to achieve this goal, oestrus synchronization is probably the most popular practice, used to improve conception rates of out-of-season ewes.
Precise synchronization of oestrus in cycling ewes or induction of oestrus in seasonally anoestrus ewes has been achieved with the use of intravaginal sponges impregnated with progestagens (medroxyprogester-one acetate, MAP, ¯uorogest(medroxyprogester-one acetate, FGA and melengestrol acetate, MGA), followed by pregnant mare serum gonadotrophin (PMSG) (Boland et al., 1981; Smith et al., 1981; Alifakiotis et al., 1982; Jabbar et al., 1994; Quispe et al., 1994; Powell et al., 1996). However, fertility of progestagen-treated anoestrus ewes has been variable, ranging from 22 to 70%. This variation has been attributed to various factors including the treatment regime, the stage of anoestrus period when treatment was initiated, ewe *Corresponding author.
breed, nutrition and lactation of ewes, seasonal changes in ram fertility, time of insemination or a combination of all the above factors (Hulet and Storm-shak, 1972; Laster and Glimp, 1976; Colas, 1979; Robinson, 1979; Echternkamp and Lunstra, 1979; Echternkamp, 1982; Romano et al., 1996). Acceptable fertility has been achieved with insemination at a predetermined time following synchronization with progestagens plus PMSG and/or hHG (Dziuk et al., 1972).
The aim of the present investigation has been to determine the optimum time of insemination in anoes-trus indigenous Greek ewes, following synchroniza-tion with MAP-impregnated intravaginal sponges and PMSG during the non-breeding season (April±July).
2. Materials and methods
2.1. Experimental animals and hormonal treatment
The present study has been conducted in Northern Greece during the non-breeding season (April±July) of indigenous Greek ewes over two consecutive years (1990±1991).
A total of 2567 indigenous Greek ewes, 507 of Chios breed (Group 1) 1236 of Vlachiki breed (Group 2), and 824 crossbred (VlachikiChios) (Group 3) were treated with intravaginal sponges impregnated with 60 mg medroxyprogesterone acetate (MAP; Ver-amix, Upjohn/Veterin, Greece). The sponges remained in situ for 14 days. All ewes were checked twice daily (a.m. and p.m.) to ensure that sponges remained in place during the treatment period. Ewes that had lost their sponge were noted and the sponge was immediately replaced. All ewes were injected with 500 IU PMSG (Intergonan, Intervet) im, at the time of sponge withdrawal. Oestrus synchronization was carried out in groups of 60 ewes so that applica-tion of arti®cial inseminaapplica-tion could be facilitated.
2.2. Time of the insemination
Within each group, the ewes were randomly divided into three subgroups (I±III) and submitted to a double blind arti®cial insemination 48 and 60 h (subgroup I), 60 and 72 h (subgroup II) and 48 and 72 h (subgroup III) after sponge withdrawal.
2.3. Semen collection and processing
Semen from Chios breed rams with excellent his-tory of fertility was collected using an arti®cial vagina. Each ejaculate was evaluated for volume, wave moti-lity and concentration. Only ejaculates with volume of more than 0.5 ml, good wave motility and a minimum concentration of 2:5109 spermatozoa/ml were used. The ejaculates from each ram were mixed, pooled and maintained at 308C. They were then diluted in one step with pasteurized skimmed cow's milk, containing 1000 IU of sodium G penicillin and 1000mg streptomycin sulfate per ml of diluent. The mixture was packed in 0.25 ml French mini straws, each containing 250ÿ300106 spermatozoa. After packaging, the straws were cooled at 158C and were maintained at this temperature by means of an ampoule containing glacial acetic acid until the time of insemination.
2.4. Arti®cial insemination
Cervical inseminations were performed on a breed-ing rack by liftbreed-ing the hindquarters of the ewe over the top rail while the front legs remained standing on the ground. Each ewe was inseminated within 4 h after semen preparation, with one 0.25 ml French mini straw containing 250ÿ300106 spermatozoa. The semen was deposited into the external os of the ®rst cervical fold, using a speculum ®tted with an internal light source.
2.5. Lambing records and statistical analysis
Fertility of inseminated ewes was expressed as lambing percentage. The fertility results were com-pared between groups using Chi-square analysis. Sig-ni®cance was taken at the level of P<0:001 (Steel and Torrie, 1980).
3. Results
than that of the ewes of Vlachiki breed (38.2%) and the crossbred (VlachikiChios) (47.6%).
Lambing rate results after a ®xed-time double insemination in anoestrus ewes treated with MAP-impregnated sponges and PMSG at various times after sponge removal are shown in Table 2.
When the ®xed-time double insemination was per-formed 48 and 60 h after sponge withdrawal, the lambing rate in ewes of Vlachiki breed (57.0%) was signi®cantly higher (P<0:001) than that of Chios breed ewes (38.8%) and in ewes of VlachikiChios breed (28.9%). On the contrary, when the ®xed-time double insemination carried out at 60 and 72 h or at 48 and 72 h after sponge removal, the lambing rates in the ewes of Vlachiki breed (31.2 and 25.0%, respec-tively) were signi®cantly lower (P<0:001) than those of the ewes of Chios (54.5 and 63.7%, respec-tively) and VlachikiChios breed (51.8 and 60.7%, respectively).
4. Discussion
The results of the present investigation indicate a marked discrepancy of the optimum time for ®xed AI in Chios, ChiosVlachiki and Vlachiki ewes, which are dif®cult to explain since there are contra-dictable references from investigators all round the world.
In anoestrus, even with a high degree of stimulation accompanied by a high ovulation rate, it is not possible to achieve an acceptable lambing rate approaching 50%. The biology of the lactating ewe and the factors Ð environmental, physical, physiological and endo-crinological Ð which in¯uence the establishment of pregnancy, are considered complicated (Robinson, 1988). The lower fertility of ewes at the ®rst oestrus, induced with progestagen-PMSG treatment, is caused by many factors including impairment of luteinizing hormone secretion, sperm transport and fertilization (Tempest and Minter, 1987).
Seasonal effects on reproduction of progestagen-PMSG treated ewes appear to be mediated through pituitary gonadotropin secretion with breed differ-ences as to the time and/or intensity of seasonal effects (Echternkamp, 1982). The interval from the onset of oestrus to preovulatory plasma LH-peak was shorter for the out-of-season Romney ewes than for the cyclic ones (Smith, 1977). In a study with out-of-season Merino ewes, the LH-peak concentration was mea-sured approximately 38 h after sponge withdrawal (Robinson, 1988).
It has been previously reported that oestrus and ovulation are highly predictable within a homoge-neous group of oestrus-synchronized ewes (Robinson
Table 1
Overall lambing percentage in the ewes of Chios breed, Vlachiki breed and their crossbred (VlachikiChios)
Genotype Number of ewes Lambing percentage (%) Inseminated Lambed
Chios 507 269 53.1a Vlachiki 1236 472 38.2b VlachikiChios 824 392 47.6a
Total 2567 1133 44.1
a,bValues with different superscripts in the same column are
different (P<0:001).
Table 2
Breed and time of arti®cial insemination effects on lambing rate of anoestrus ewes treated with MAP-impregnated sponges and PMSG Genotype Time of artificial inseminationc
48 and 60 h 60 and 72 h 48 and 72 h Chios 38.8b(62/160)d 54.5a(84/154) 63.7a(123/193)
Vlachiki 57.0a(245/430) 31.2b(128/410) 25.0b(99/396)
VlachikiChios 28.9b(75/260) 51.8a(147/284) 60.7a(170/280)
Overall 44.9 (382/850) 42.3 (359/848) 45.1 (392/869)
a,bValues with different superscripts in the same column are different (P<0:001).
and Smith, 1967; Evans and Robinson, 1980; Robinson et al., 1987), with a close relationship between the onset of oestrus and the LH-surge (Evans and Robinson, 1980; Pearce and Robinson, 1985; Robinson et al., 1987). On the other hand it has been reported that the onset of oestrus can be variable (Maxwell et al., 1986). Results of a previous investi-gation (Amir and Schindler, 1972) indicated that even a time interval of 40 h from insemination to the end of oestrus did not in¯uence the conception rate. Since the end of oestrus coincides on the average with ovulation, this time interval is indicative of the duration of the fertile life of the spermatozoa.
Precise time interval of sponge withdrawal to oes-trus is also dif®cult to determine and can be varied as in Merino ewes referred between 24 and 48 h (Max-well and Barnes, 1986), or 59.7 h (Max(Max-well, 1986b) and in Galway ewes 58±78 h (Quirke et al., 1979). In Chios ewes, this interval was 45 h (Brozos et al., 1999), but all the above mentioned experiments were conducted during breeding season.
The optimum time for insemination varies among different ewe breeds. It has been reported that for LeicesterSwaledale ewes the optimum time for insemination is between 54 and 60 h after sponge withdrawal (Findlater et al., 1991), while for Merino ewes it was suggested to be between 60 and 72 h post sponge withdrawal, although the mean time of ovula-tion is the same in these two breeds (Maxwell, 1986a; Eppleston and Roberts, 1986). For Suffolk ewes, it has been reported that the optimum time for insemi-nation is 48 h after sponge removal (Fukui et al., 1989; Findlater et al., 1991), while in Border LeicesterScottish Blackface ewes, high conception rates were achieved applying AI 54±58 h after sponge withdrawal (Aitken et al., 1990).
The use of 500 IU PMSG on sponge withdrawal was reported to have improved fertility (Hackett and Wolynetz, 1982; Langford et al., 1983) but the response to this dose among different breeds can be varied. Chios breed is characterized by having ovarian activity almost all year around while VlachikiChios and Vlachiki breed are seasonally breeding ewes (Vosniakou, 1983). Thus, the dose of 500 IU might not have been suf®cient to stimulate follicular devel-opment in some Vlachiki and VlachikiChios ewes, leading to lower overall conception rates for these two breeds.
Finally, it is well known that stress can delay or block the appearance of oestrus and even reduce fertility by impairing the cervical transport of sperm (Marinov, 1984; Ehnert and Moberg, 1991). Although all ewes of the present investigation were handled similarly, the fact that ewes of Chios breed are more accustomed and tolerant in handling than the other two breeds used, which could be of some importance.
From the results of the present investigation, it can be concluded that conception rate after progestagen treatment in anoestrus Chios ewes is better than that of VlachikiChios breed with the latter being better than that of Vlachiki breed. Using ®xed time for AI, better conception rate is obtained when applied 48 and 72 h after sponge withdrawal for Chios and ChiosVlachiki breeds while for Vlachiki breed better conception rate is obtained when ®xed AI is applied 48 and 60 h after sponge withdrawal.
References
Aitken, R.P., Wallace, J.M., Robinson, J.J., 1990. A note on conception rates and litter sizes following the intrauterine insemination of ewes at an induced oestrus during seasonal anoestrus. Anim. Prod. 50, 379±382.
Alifakiotis, T., Michailidis, I., Gavrilidis, G., 1982. Induced breeding in anoestrus milking ewes of dairy breeds: comparison of norgestomet, medroxyprogestertone and ¯uorogestone in two regimes of PMSG. Theriogenology 17, 603±610. Amir, D., Schindler, H., 1972. The conception rate of ewes after
arti®cial insemination at different times during oestrus. J. Reprod. Fertil. 28, 261±264.
Boland, M.P., Crosby, T.F., Gordon, I., 1981. Induction of pregnancy in lactating anoestrus ewes. J. Agric. Sci. (Camb.) 97, 465±467.
Brozos, C.N., Saratsis, Ph., Boscos, C., Kyriakis, S.C., Alexopou-los, C., 1999. The effect of bovine somatotropin (bST) administration on reproduction, progesterone concentration during lactation and LH-secretion during oestrus, in dairy ewes. Anim. Reprod. Sci. 56, 177±187.
Colas, G., 1979. Fertility in the ewe after arti®cial insemination with fresh and frozen semen at the induced oestrus, and in¯uence of the photoperiod on the quality of the semen. Livest. Prod. Sci. 6, 153±166.
Dziuk, P.J., Lewis, J.M., Graham, E.F., Moyer, R.H., 1972. Comparison between natural service and arti®cial insemination with fresh or frozen sperm at an appointed time in the ewe. J. Anim. Sci. 35, 572±575.
Echternkamp, S.E., Lunstra, D.D., 1979. Causes for decreased fertility in out-of-season mated ewes. Theriogenology 10, 65±70. Ehnert, K., Moberg, G.P., 1991. Disruption of estrous behavior in ewes by dexamethazone or management -related stress. J. Anim. Sci. 69, 2988±2994.
Eppleston, J., Roberts, E.M., 1986. The effects of progestagen, PMSG and time of insemination on fertility in ewes following intrauterine insemination with frozen semen. Aust. Vet. J. 63, 124±125.
Evans, G., Robinson, T.J., 1980. The control of fertility in sheep: endocrine and ovarian responses to progestagen-PMSG treat-ment in the breeding season and anoestrus. J. Agric. Sci. (Camb.) 94, 69±88.
Findlater, R.C.F., Hareign, W., Curnock, R.M., Beck, N.F., 1991. Evaluation of intrauterine insemination of sheep with frozen semen effects of time of insemination and semen dose on conception rates. Anim. Prod. 53, 89±96.
Fukui, Y., Akaike, M., Anzai, H., Ono, H., 1989. Effect of timing of injection with pregnant mare serum gonadotrophin on ®xed-time arti®tial insemination of seasonally anoestrus ewes. J. Agric. Sci. (Camb.) 113, 361±364.
Hackett, A.J., Wolynetz, M.S., 1982. Effect of PMSG on the reproductive performance of totally con®ned ewes bred at synchronized oestrus. Theriogenology 17, 215±221.
Hulet, C.V., Stormshak, F., 1972. Some factors affecting response of anestrus ewes to hormone treatment. J. Anim. Sci. 34, 1011± 1019.
Jabbar, G., Umberger, S.H., Lewis, G.S., 1994. Melengestrol acetate and norgestomet for the induction of synchronized oestrus in seasonally anovular ewes. J. Anim. Sci. 72, 3040± 3054.
Langford, G.A., Marcus, G.J., Batra, T.R., 1983. Seasonal effects of PMSG and number of inseminations on fertility of progestagen treated sheep. J. Anim. Sci. 57, 307±312.
Laster, D.B., Glimp, H.A., 1976. In¯uence of breed on response to exogenous hormones in estrous and anestrus ewes. J. Anim. Sci. 42, 893±900.
Marinov, M.F., 1984. Effect of the type of restraint of sheep during arti®cial insemination on their fertility. Vet. Med. Nauki 21, 74±78.
Maxwell, W.M.C., 1986a. Arti®cial insemination of ewes with frozen-thawed semen at a synchronized oestrus. Effect of dose of spermatozoa and site of insemination on fertility. Anim. Reprod. Sci. 10, 309±316.
Maxwell, W.M.C., 1986b. Arti®cial insemination of ewes with frozen-thawed semen at a synchronized oestrus. Effect of time of onset of oestrus, ovulation and insemination on fertility. Anim. Reprod. Sci. 10, 301±308.
Maxwell, W.M.C., Barnes, D.R., 1986. Induction of oestrus in ewes using a controlled internal drug release device and PMSG. J. Agric. Sci. (Camb.) 106, 201±203.
Maxwell, W.M.C., Hunton, J.R., Ryan, J.P., Hood, G., 1986. Effect of GnRH on time of ovulation in superovulated Merino ewes.
In: Proceedings of the 18th Conference on the Soc. Reprod. Biol. (Aust. Soc. Reprod. Biol.: Canberra) p. 20.
Powell, M.R., Kaps, M., Lamberson, W.R., Keisler, D.H., 1996. Use of melengestrol acetate-based treatments to induce and synchronize oestrus in seasonally anoestrus ewes. J. Anim. Sci. 74, 2292±22302.
Pearce, D.T., Robinson, T.J., 1985. Plasma progesterone concen-trations, ovarian and endocrinological responses and sperm transport in ewes with synchronized oestrus. J. Reprod. Fertil. 75, 49±62.
Quirke, J.F., Jennings, J.J., Hanrahan, J.P., Gosling, J.P., 1979. Oestrus, time of ovulation, ovulation rate and conception rate in progestagen-treated ewes given GnRH, GnRH analogues and gonadotrophins. J. Reprod. Fertil. 56, 479±488.
Quispe, T., Zarco, L., Valencia, J., Ortiz, A., 1994. Estrus synchronization with melengestrol acetate in cyclic ewes, insemination with fresh or frozen semen during the ®rst or second oestrus post treatment. Theriogenology 41, 1385±1392. Robinson, T.J., 1979. Controlled breeding of sheep and goat. In: Tomes, G.J., Robertson, D.E., Lightfoot, R.L., Haresign, N. (Eds.), Sheep Breeding. Betterworths, London, pp. 423±437. Robinson, T.J., 1988. Controlled sheep breeding: update 1980±
1985. Aust. J. Biol. Sci. 41, 1±13.
Robinson, T.J., Scaramuzzi, R.J., Smith, C.A., 1987. The time of mating and of LH-release and subsequent fertility of anoestrus Border LeicesterMerino ewes treated with progestagen and
pregnant mare serum gonadotropin. Anim. Reprod. Sci. 13, 23± 36.
Robinson, T.J., Smith, J.F., 1967. The time of ovulation after withdrawal of SC-9880-impregnated intravaginal sponges from cyclic Merino ewes. In: Robinson, J. (Ed.), The Control of the Ovarian Cycle in the Sheep. Sydney University Press, Sydney, pp. 158±168.
Romano, J.E., Rodas, E., Ferreira, A., Lago, I., Benech, A., 1996. Effects of progestagen, PMSG and arti®cial insemination time on fertility and proli®cacy in Corriedale ewes. Small Rumin. Res. 23, 157±162.
Smith, J.F., 1977. Estrus, ovulation and conception following timed insemination in Romney ewes treated with progestagen and gonadotropins. Theriogenology 7, 63±72.
Smith, P.A., Boland, M.P., Gordon, I., 1981. Effect of type of intravaginal progestagen on the outcome of ®xed-time arti®cial insemination. J. Agric. Sci. (Camb.) 96, 243±245.
Steel, R.G.D., Torrie, J.M., 1980. Principles and Procedures in Statistics. McGraw-Hill, New York, 1980.
Tempest, W.W., Minter, C.M., 1987. Synchronized breeding and lambing. In: Fayes, M., Marai, M., Owent, J.B. (Eds.), New Techniques in Sheep Production. Butterworth, London, pp. 221± 239.
