MERINO EWE LAMBING AFTER STIMULATION WITH PROSTAGLANDIN AND PMSG AND INSEMINATION WITH SPERM OF GREY

Aim. To determine the effectiveness of a two-time treatment with prostaglandin F-2α (PGF2α) followed by the injection of pregnant mare serum gonadotropin (PMSG) for stimulation of sexual activity in sheep in the second half of the estrous season in the scheme of increasing the lambing rate. Methods. Thirty-seven 2.5-yearold ewes of the Askanian Merino breed, which were kept together from the age of one year, were used. In September 2018, 28 animals of this group lambed and had sucklings until November 20. Hormonal stimulation was started on December 7, 2018 by injecting all ewes with 1 ml of synthetic PGF2α and 1 ml of Prozerin. The second injection of PGF2α was given 10 days after the fi rst one. Simultaneously with the second treatment with PGF2α, the animals were injected with 500 IE of PMSG (Sergon, Czech Republic) and 2.5 ml of SuperAminoC (South Korea). The presence of estrus was not detected, and the ewes were forcibly inseminated with freshly obtained sperm of 4 grey rams of the Askanian Karakul breed in the morning for three days, beginning with the fi rst day after the PMSG injection. During the fi rst treatment with PGF2α and 3 days before the second one, the genitals of 12 ewes were sonographically examined. Results. In total, 45.9 % of stimulated animals lambed; the total fecundity was 75.7 %, and the average prolifi cacy – 1.65 ± 0.18 lambs per ewe. Among the animals, which before the experiment had the sucklings, 57.1 % lambed, among those, who did not have the suckling, – 11.1 %. After using the sperm of ram 17314, 77.8 % of ewes lambed, for rams 73697, 73792 and 73796 – 30.0, 44.4 and 33.3 %, respectively. Among the 19 lambs analyzed by wool color, 10 lambs were completely black, 9 were black with white spots. Among the spotted lambs, the color on the back, forearms and hips of one lamb had an additional shade, close to gray. Ultrasound testing showed that the ewes, which before the experiment had sucklings, showed better genital reactivity. According to the distribution of lambs by wool color, an assumption about possible partial incompatibility of the genotypes of Askanian Merino ewes and grey Karakul sheep was made that requires further study. Conclusions. The scheme of stimulating sexual activity based on two-time treatment with PGF2α followed by an injection of PMSG is capable of ensuring lambing of more than 70 % of stimulated sheep with the prolifi cacy of 1.65 lambs per ewe. A factor, increasing the effectiveness of the scheme, is the presence of sucking lambs of ewes before the stimulation starts.


INTRODUCTION
The increase in lambing is one of the ways to increase the effi ciency of sheep breeding. However, the majority of sheep, kept in middle latitudes, demonstrate seasonality of reproduction. To achieve insemination at desired time, ewes are subject to hormonal or technological stimulation.
At present, the main scheme of hormonal initiation of estrus in ewes both during the anoestrous and mating season envisages subjecting animals to a longterm effect of gestagens with subsequent stimulation of folliculogenesis using gonadotropins. The most convenient and wide-spread form of gestagenization is the application of vaginal pessaries or CIDR devices, containing synthesized or natural gestagens. A considerable advantage of the mentioned devices is the fact that changing the hormone content in them, one may control the concentration of gestagens in the organism of animals. Also, the application of pessaries and CIDR allows initiating and completing the treatment of many animals at the same time and without any consideration of the presence and stage of oestrous cycle [1]. But long-term application of intravaginal devices may provoke the development of opportunistic bacteria in vagina [2] which is not overcome even using antibiotics. Also, the application of pessaries may decrease sexual attraction of ewes for rams [3], and decrease the basal level of luteinizing hormone [4]. The mentioned drawbacks force for the search of alternative schemes of stimulation.
Gestagens in the organism beyond the pregnancy period fulfi ll two main functions. Firstly, they prepare cellular structures of ovaries [5] and sensibilize some links of hypothalamus [6][7][8] to the effect of other hormones and substances. Secondly, gestagens inhibit early development of antral follicles [9,10]. Therefore, to create the conditions for the growth of follicles, which are then to ovulate, the negative impact of gestagens should be removed at a certain time, in particular, via decreasing their concentrations. When intravaginal devices are used, the drop in the content of gestagens is provoked by their mechanic removal. But during the mating season, the ovaries of most ewes have periodic corpora lutea, which are the source of endogenous gestagen progesterone, and a rapid decrease in the content of this hormone should be achieved by measures, terminating the progesterone synthesis with their own structures. The latter is implemented with natural and synthetic analogues of prostaglandins of group F-2 alpha (PGF2α), which initiate the lysis of corpora lutea.
At the same time, the presence of corpora lutea in the ovaries of ewes during the mating period allows avoiding the application of exogenous gestagens, and thus avoiding the use of vaginal sponges and eliminate the related negative impact on the vagina. However, it sets certain requirements to the quality of stimulation.
The effective scheme of applying prostaglandins to ewes has not been suffi ciently determined yet. The factors, impacting the fi nal results, are the frequency and dosage of drug administration, additional treatment with other hormones, degree of functionality of the available corpora lutea, etc. For instance, a single treatment of ewes with PGF2α resulted in lower indices of lambing compared to the animals, treated with vaginal pessaries and gonadotropins [11]. The main drawback of a single treatment with prostaglandin is the fact that it should be connected to a certain stage of the estrous cycle, which does not allow for frontal treatment of animals. To overcome the latter, the number of PGF2α injections is increased to two, with the interval of 7-11 days between them. However, even two-time treatment does not guarantee suffi cient and stable results. Thus, although two-time injections of the synthetic analogues of PGF2α to ewes with the interval of 7 days facilitated the accelerated development of follicles, as a result they decreased the number of ovulations, increased the embryonic mortality and conditioned reliably lower indices of conception, prolifi cacy and fecundity compared to the indices of animals with spontaneous estrus [12]. In another experiment the index of Merino sheep lambing after two-time injections of prostaglandin with the interval of 7 days in the mating season was also low (26-31 %), reliably lower than the index of the scheme of intravaginal pessaries and gonadotropins [13]. At the same time, the indices of lambing for coarse-wool sheep after two treatments with PGF2α were rather high -73.3-75.9 % [14]. The drawbacks of PGF2α application are not conditioned by the variant of its production. The comparison of the effi ciency of two-time injections of different synthetic analogues of prostaglandin -dinoprost and cloprostenol -did not demonstrate reliable differences in the general indices of lambing [15].
The effectiveness of PGF2α application may be increased, if the stimulation scheme is supplemented with other hormones and substances. For instance, the treatment of sheep with intravaginal pessaries prior to the single injection of cloprostenol improved the manifestation of estrus and the conception rate of animals [16]. In total, the treatment of sheep with gonadotropin-releasing-hormone (Gn-RH) fi ve days prior to the injection of PGF2α increased the fecundity and prolificacy compared to the control non-stimulated animals. [17]. However, as stated above, the application of intravaginal devices may lead to negative adverse events, and corpora lutea, formed under the effect of Gn-RH are not always suffi ciently functional [18]. In addition, the schemes of stimulating sexual activity, using prostaglandin, require enhancement.
Naturally, after the decrease in the content of progesterone the development of follicles is controlled by endogenous factors [19,20]. However, sometimes due to some reasons the effect of the impact of these fac-tors gets weaker and they become incapable of stimulating and supporting the development of follicles. It was demonstrated that one of the reasons for the latter may be a month of the year [21]. The inability of endogenous factors to support the growth of follicles conditions the need to apply additional stimulating measures. This measure may be additional treatment of animals using follicle-stimulating substances, including pregnant mare serum gonadotropin (PMSG). However, the data on the effectiveness of double treatment using PGF2α with additional treatment with PMSG in the schemes of stimulating sexual activity of sheep in the second half of the mating season are insuffi cient.
In technological schemes to increase the number of lambings, the interval between lambing and subsequent insemination is maximally reduced to such that would not aggravate the development of the weaned lambs. But it raises the question whether ewes are capable to restore after weaning suffi ciently to get pregnant, and how well the scheme using PGF2α is suitable for stimulation of sexual activity in such animals. The studies of Ungerfeld R. and Sanchez-Davila F. (2012) demonstrated the ability of ewes to get pregnant after reducing the traditional period of sucking, but here the traditional scheme with gestagens and PMSG was used for stimulation [22].
The main aim of our experiment was to determine the effectiveness of PGF2α application in stimulating sexual activity of sheep in the second half of estrous season. A scheme with two-times injections of PGF2α with the interval of 10 days and additional treatment with PMSG was selected for the study. Another issue was to compare the effectiveness of applying the suggested scheme to ewes, which had or had not sucking lambs prior to stimulation. The effectiveness of stimulation was estimated by the results of subsequent lambing of animals.

MATERIALS AND METHODS
Animals. Thirty-seven 2.5-year-old ewes of the Askanian Merino breed were used in the experiment. The animals of this breed are remarkable for seasonal type of reproduction with the start of natural manifestation of estrus in the third decade of August -fi rst half of September, and disappearance of ovulation cases in March-April [23]. The ewes belonged to and were kept at the experimental farm «Askania-Nova» of the Institute of Animal Husbandry «Askania-Nova», located on 46°27´ north latitude and 33°52´ east longitude. The animals were kept together since the age of one year and composed a group, used to study the effectiveness of the scheme of increasing the frequency of lambing. Within the latter, in September 2018, 28 ewes lambed and till November 27 had sucking lambs.
Scheme of manipulations. The stimulation was started on December 07, 2018 with an intramuscular injection of 250 mg of the synthetic analogue of PGF2α (cloprostenol, Bioestrophan, Ukraine) and 1 ml of Prozerin (0.5 %, Ukraine). On December 17 the second injection of PGF2α was done in the same dose, with simultaneous administration of 500 IU of PMSG (Sergon, Czech Republic) and 2.5 ml of SuperAmino-C (South Korea). The latter is a mixture of free aminoacids and group B vitamins. The presence of a heat in the stimulated ewes was not tested, and they were forcibly inseminated in the morning for three consecutive days, beginning with the fi rst day after the PMSG injection. On December 7 and 14, the ewes were subject to ultrasound scanning with the determination of the values of functionality indices of their genitals. Freshly obtained sperm of 4 grey adult rams of the Askanian Karakul breed was used for insemination.
Used data. The effectiveness of stimulation was determined by the indices of fertility (F%), prolifi cacy (Fm%) and fecundity (Pf). F% of animals was determined by the division of the number of lambed ewes by the total number of animals and then multiplication by 100. Fm% was determined by the division of the number of all the obtained lambs by the total number of animals and subsequent multiplication by 100. Pf was determined by the division of the total number of obtained lambs by the number of ewes, which lambed.
The indices of the functionality of the genitals were determined using a manual US-device with the probe frequency of 5 MHz. The study envisaged the determination of degrees of uterine friability (UF, from 0 to 3 points), ovarian proliferation (OP, from 0 to 3 points) and folliculogenesis (FG, from 0 to 3 points). The values of all the indices were determined subjectively. UF index depended on the gradient of echogenicity of uterine horns. The lesser difference there was between the echogenicity of different areas of the horn, the larger values of UF were. The index of OP was estimated according to the number of observed follicles and the ratio of their diameter. The less the ratio of diameters of large and small follicles differed from 1 : 1, the smaller OP was. The index of FG was estimated in accordance to the diameter of the group of the largest follicles (0, 0-1, 1-3, and over 3 mm).
The statistical processing of the data was done according to common ANOVA-algorithms, using the mathematical apparatus of Excel, Microsoft Offi ce package, the probability (p) of difference in indices were defi ned by Student's criterion (t d ).

RESULTS
In total, 45.9 % of ewes lambed. To fi nd out possible reasons of low total effectiveness, the indices were analyzed with the consideration of either the presence of prior sucklings of animals (groups 1 and 2) or the sire, whose sperm was used during insemination (groups 3-6) ( Table 1). The index F% of animals which had sucklings prior to the experiment (group 1) was reliably higher than the index of ewes without prior sucklings (group 2). Fm% was also higher for animals with prior sucklings. No reliable difference was revealed between groups for Pf. Therefore, the presence of prior sucklings of animals conditioned the difference in the quality of reacting to the stimulation. It is noteworthy that previously farrow ewes had unreliably larger bodyweight and thus insuffi cient fatness could not be a reason of their infertility.
While grouping the data of lambing according to sire, the highest indices F% and Fm% were seen for the use of sperm of ram 17314 (group 3). When the sperm of three other ram was used, the F% index was much worse. Reliable difference in terms of index Pf was observed only between rams 17314 and 73792. As the average index of the activity of the used sperm, calculated by averaging the values for three days when insemination was performed, demonstrated almost no differences between ram, the quality of sperm could not have been the reason in the conception of ewes. It should be noted that rams, whose sperm was used to inseminate the stimulated ewes, had been used in the previous autumn mating season, after which they had two months to get restored. Here, the F% index for Karakul ewes in autumn mating campaigns of 2017 and 2018 using ram 17314 was 67 and 43 %, 73697 -50 and 42 %, 73792 -67 and 60 %, and 73796 -100 and 75 %. Thus, the difference in the insemination capability between rams during the mating season did not coincide with that in our experiment.
Among 19 obtained lambs, evaluated by the wool color, 10 animals were completely black. Nine had small white spots, mainly localized on the forehead, around eyes, on the lower jaw, extremities and tail. Among the spotted lambs, one animal had additional almost gray color on the back, forearms and hips. Barely visible lighter color in the same places was revealed in two more lambs out of the spotted group. The distribution of lambs by color (black/spotted) in terms of inseminators was as follows: ram 7314 -37/63, 73697 -67/33, 73792 -75/25, 73796 -40/60. Ram 17314 was the father of a lamb with gray wool and a lamb with lighter color. Ram 73796 was the father of one lamb with slightly lighter color of the wool. The wool around eyes and on the forehead became brown after a month and a half in two black lambs, obtained from ram 17314. Thus, only one lamb out of 19 had visible features of gray wool color. Its father was a ram, after the use of whose sperm the F% index was the highest.
The results of US-scanning of ewe genitals are presented in Table 2. A total of 12 animals were tested, and their data were grouped according to the presence of either prior sucklings (group 1 and 2) or lambing after Table 1. Reproduction indices of ewes Notes: 1)* -ram sperm activity, averaged by the activity of three days, during which insemination was done.
2) Here and further on, the values in one column with different subscripts differ with the probability of p < 0.05.

Group
Presence of previous sucklings (+/-)/sire  3 and 4). At the beginning of stimulation, the indices of UF and OP for animals with prior sucklings (group 1) were unreliably higher than similar indices for animals without prior sucklings (group 2) which demonstrates higher functionality of the genitals of animals in group 1. Seven days later, the difference between the mentioned indices of UF and OP became reliable, but here the direction of changes in these indices in groups 1 and 2 was opposite. The FG index for animals of group 1 prior to the second injection of PGF2α decreased unreliably and had almost no changes in group 2. The difference between the indices of the fi rst and second testing for animals of group 1 was also higher for a similar difference in group 2. Three out of four scanned animals with prior sucklings lambed after the stimulation, while no ewe lambed among those that had not had prior sucklings.
The grouping of animals by the fact of subsequent lambing demonstrated that at the beginning of stimulation the indices of UF and OP for animals, which lambed after the stimulation (group 3) were reliably higher than the indices of animals which remained nulliparous (group 4). Seven days later these indices for animals of group 3 decreased. The UF index increased for ewes of group 4 after a week, and that of OP almost unchanged. The difference for FG was unreliable both while comparing the indices for groups and the scanning dates.
Therefore, US-scanning demonstrated the difference in absolute values and the dynamics of changes in UF and OP indices: 1) between the ewes, which prevously had sucking lambs, and those that had not; and 2) between ewes which became pregnant after the stimulation and those that remained farrow. The largest differ-ence between the absolute values of the indices of the fi rst and second testing demonstrates better response of the genitals of animals in group 1 compared to that of ewes in group 2, and the ewes in group 3 compared to the animals of group 4, which may explain the difference, observed in the fertility of the stimulated animals.

DISCUSSION
Though the total fertility in the experiment was unsatisfactory, the data of the lambing of animals, inseminated with the sperm of ram 17314, demonstrate potentially suffi cient effectiveness of the used scheme of stimulation. In our opinion, the main reason of low the total fertility may be the month of conducting an experiment -December, which is a part of the second half of oestrous season for Askanian Merino breed. It is known that the sensitivity of cellular structures of ovaries is lower at the end of the oestrous cycle [7]. The results of US-scanning in our experiment demonstrated that the genitals of animals which remained farrow had decreased functionality at the beginning of the experiment and worse response to the fi rst treatment with prostaglandin. The second injection and additional stimulation with gonadotropin could not compensate unsatisfactory response to the fi rst administration of PGF2α.
A connection, determined between the effectiveness of stimulation and presence of prior sucklings, is signifi cant for practice. It is believed that the period of sucking leads to lactation anestrus in ewes. However, it is known that if the delivery takes place during the natural oestrous period, the lactation anestrus may not be evident, and some ewes are capable of getting pregnant even having a sucking lamb [22]. The results of our experiment demonstrate that during the oestrous The result of lambing of the experimental ewes and the revealed dependence in the effi ciency of stimulation with the presence of previous sucklings gives grounds for assumption that the scheme of hormonal stimulation of sexual activity in sheep using two-time treatment with PGF2α and subsequent injection of PMSG in the second half of the oestrous period is reasonable for application only to the animals which had sucking lambs prior to the stimulation.
The difference in fertility of ewes after insemination with sperm of different rams and the specifi cities of dividing lambs by wool color allows assuming one more possible reason of low total effi ciency of stimulation. This reason may be the incompatibility of genotypes of grey Karakul rams and Merino ewes. It is known that grey color of wool is determined in Karakul sheep by two alleles -dominant E D of Extension-locus, related to chromosome 14 [24,25], and dominant letal roan (Rh Rh ) of Roan-locus [26]. Rh Rh epistatically inhibits E D [27]. As the used rams were heterozygous by allele Rh Rh , half of all the lambs in our experiment should have had gray wool, which was not observed. The latter may be explained by the death of embryos on early stages. It may be assumed that as Merino and Karakul breeds are genetically rather distant, the localization or mechanism of activation for some genes may not coincide in them. In this case of insuffi cient functionality of the chromosome, coming from grey Karakul, the genes on its homologue from Merino ewe are incapable of activating properly, which may condition the death of embryos. As for the presence of some animals with lighter wool among the lambs, in our opinion it may be explained by two factors. Firstly, the genotype of some gray rams may have some gene combinations, whose functioning eliminates the negative impact of allele Rh Rh and promotes survival of some embryos. Secondly, there are data, proving that in case of the genotype, homo/heterozygous by allele Е + and absence of letal allele Rh Rh the manifestation of gray color in Karakul sheep may be initiated by the modulating effect of the locus Agouti [26].
At this point, these are merely our assumptions, as the number of articles on the study of genetic reasons of Karakul sheep lethality is limited. Recent studies demonstrated that in a promotor region of melanocortin receptor gene MC1R, Karakul sheep have 3 points of single-nucleotide polymorphism (SNP) and an insertion of 26 nucleotides. The authors relate the lethality among Karakul sheep to the presence of the nucleotide insertion [28]. GWAS-analysis detected several SNPs, associated to sublethality, in chromosomes 3, 5 and 8 in the animals of Swakara breed, which originate from Central Asian Karakul sheep [29]. Here, three SNPs were related to chromosome 3, with which gene IGF1 is associated in sheep, the beginning of expression of which in sheep embryos was registered at a one-cell stage [30]. It should be noted that gray color in sheep is often associated with the increase in embryo losses. For instance, as for sheep of Wensleydale breed, in which black color is a recessive feature, after the insemination of white ewes with the sperm of gray rams, 42.8 % of animals lost their pregnancy. When using gray rams with black ewes the level of abortions decreased to 25 % and when black rams were used with white ewes -to 16.7 % [31]. The lambs of Garole breed, which demonstrate features of being roan, perish up till day 4 [32].
The available literature sources contain only several mentions of cross-breeding of Merino sheep and Karakul rams, but there are no accurate data about the wool color of the used rams or reproduction indices. Taking into consideration the results of lambing, the specifi cities of cross-breeding of grey Karakul rams and Merino ewes requires additional studies.

All the manipulations with animals were done in compliance with the provisions of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientifi c Purposes
Ключевые слова: овца, Асканийская Тонкорунная порода, серый Каракуль, гормональная стимуляция, простагландин Ф-2α, ГСЖК