It is clear that lambing rate depends mainly on the ovulation rate ascribed to a given breed (1, 2, 11, 27), influence of gonadotrophins (9, 15), oxytocin (3), metabolic hormones as well as auto- and paracrine factors (VEGF, FGF, IGFs) (17, 21). According to Findlay et al. (7) multiple ovulation in ewes is brought about by a change in the window during which gonadotropin-dependent follicles are viable. Despite the central importance of FSH for folliculogenesis, its role in augmentation of ovulation rate (OR) is poor (7, 16). Consequently, some authors (4-6, 28) turn atten-tion to LH, which is capable of influencing the num-ber of ovulated follicles and the quality of oocyte. Sub-sequently, Spicer et al. (23) focused on the members of IGF system as follicular factors, appearing also in plasma, which exert an effect on multiple ovulation in ewes. Meanwhile, Driancourt et al. (5) suggest that LH and presumably also its counterpart hCG (human
chorionic gonadotropin) affect folliculogenesis not only by its receptors but also by the induction of paracrine mediators, including the members of epidermal growth factor (EGF) family: namely, amphiregulin, epiregulin and â-regulin. Moreover, these authors established that improvement in OR, under the influence of hCG given during anestrus or during luteal phase, is more apparent in non- prolific breeds. Furthermore, perple-xing aspects of LH action on the follicle(s) are derived from the restriction of the LH receptor (LHR) expres-sion to mural granulose cells, which can be overcome by released factors propagating LH stimulus on neigh-boring area.
Taking into account above data, the aim of the study was to define the influence of hCG on the ovulatory and prolific responses in two breeds of Polish ewes (low-prolific PLS and high prolific BCP) administe-red during the quick proceeding folliculogenesis and
Reproductive performance of PLS and BCP ewes
exposed to hCG at the follicular phase
of the estrous cycle
RYSZARD BOBOWIEC, URSZULA KOSIOR-KORZECKA, KRZYSZTOF PATKOWSKI*, TOMASZ GRUSZECKI*, EL¯BIETA TUSIÑSKA
Department of Pathophysiology, Chair of Preclinical Veterinary Sciences, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12, 20-033 Lublin, Poland
*Department of Sheep and Goat Breeding, Faculty of Biology and Animal Breeding, University of Life Sciences, Akademicka 13, 20-950 Lublin, Poland
Bobowiec R., Kosior-Korzecka U., Patkowski K., Gruszecki T., Tusiñska E.
Reproductive performance of PLS and BCP ewes exposed to hCG at the follicular phase of the estrous cycle
Summary
Ovulation rate, prolificacy, plasma IGF-I and steroid hormones concentrations were compared amoung two breeds of Polish ewes exposed to hCG. Both low (PLS) and high prolific group (BCP) ewes were separated randomly into three subgroups (10 ewes each): control animals injected with an equal volume of saline solution, once treated with hCG (300 IU, i.m.) on the 15th day of the oestrus cycle, and twice treated with 300 IU, i.m hCG on the 13th and 15th days of the oestrus cycle. Ovulation rate for once treated PLS and BCP groups (2.6 and 2.9 respectively) were always higher than in double treated groups (2.0 and 2.4 respectively). Stimulatory influence of hCG on ovulation rate was more pronounced in less prolific PLS breed than in BCP sheep. The prolificacy was augmented to a considerably greater extent in ewes exposed to double treatment with hCG, with significant differences noted only in BCP ewes. In all ewes used in this experiment, the number of ovulatory follicles rose together with the increment of plasma IGF-I concentration. The most influencal level of IGF-I on the number of ovulatory follicles following hCG treatment ranged between 28.0-31.0 ng/ml. The obtained results indicate that optimal time to induce extra ovulatory response to hCG is the beginning of estrus, whereas hCG given at the end of estrus time is effective in improving prolificacy in ewes.
the time of estrus. We were also interested in determi-ning the changes in ovarian steroids and IGF-1 plasma levels as a result of hCG treatment.
Material and methods
Animals and experimental design (fig. 1). Sixty cyclic ewes (thirty low prolific Polish Lowland Sheep (PLS) and thirty high prolific BCP sheep (the synthetic meat line: Polish Lowland Sheep × Charolaise x Berrichone du Cher × Romanov × Olkuska) were used. The ewes were 3-5 years of age and weighted 65-76 kg. The experiments were carried out in August and September during the breading season. All ewes were given two injections of 200 µg PGF2á analogue (Cloprostenol, Oestrophan, Leciva, Czech Repu-blic) at 11-day intervals to synchronize the estrous cycle. 48 h after the second PGF2á injection, at the expected estrus time, blood samples from the jugular vein were col-lected to check the efficiency of estrus synchronization by HPLC analysis of 17â-oestradiol (E2) and progesterone (P4). The results of hormonal analysis (mean E2 concen-tration: 29.30 ± 4.55 pg/ml, mean P4 concenconcen-tration: 0.53 ± 0.09 ng/ml) enabled us to recognize estrus and confirmed the efficacy of synchronization. All ewes were divided into two groups according to the breed: the 1st group PLS (n =
30) and the 2nd one BCP (n = 30). Sheep were fed with
meadow hay (0.3 kg/d/ewe), green forage (red clover, 4 kg/ d/ewe), dried beet pulp (0.3 kg/d/ewe) and oats grain (0.15 kg/d/ewe) (1.36 kg of dry matter, gross energy: 5.67 MJ/kg of dry matter, 10.88% digestible protein on a dry matter basis). All animals had free access to fresh water. Both the 1st (PLS) and the 2nd group (BCP) were separated randomly
into three subgroups: 2 × hCG twice treated with hCG (300 IU, i.m., Chorulon, Intervet) on the 13th and 15th day
of the estrous cycle (n = 10); 1 × hCG obtaining hCG once (300 IU, i.m., Chorulon, Intervet) on the 15th day of
the estrous cycle (n = 10); cont control animals injected with the equal volume of physiological salt solution (i.m.) (n = 10). On the 16th-17th day of the estrous cycle all ewes
were mated. Each day from the 12th to the 17th day of
the estrous cycle, 10-ml blood samples for the analysis of insulin-like growth factor-1 (IGF-1), E2 and P4 concentra-tions were collected from the jugular vein. The blood was centrifugated (20 min at 4°C, 1000 g) and the obtained plasma was stored at 20°C. On the 6th and 7th day of the
following estrous cycle the ovulation rate in all ewes was determined by the laparoscopy of ovaries.
Analytical procedures. Steroid hormones (E2 and P4) were analyzed by HPLC (Beckman, Gold System, Fuller-ton, CA, USA) with UV detection according to the proce-dure described before (14). Briefly, the separation of hor-mones was performed in a gradient of acetonitrile (40-100% in 20 min), whereas peak detection at 220 nm (DAD 168; Beckman). As an internal standard dehydrocholic acid was used. IGF-I level was determined using radioimmunoassay (IGF-I-D-RIA-CT; Biosource, Nivelles, Belgium).
Statistical analysis. Ovulation rate has been defined as the number of corpora lutea (CL) per ovulating ewe. Proli-ficacy has been estimated by dividing the number of lambs born alive per the number of lambing ewes (e.g. 1,5). The obtained data were analyzed with using Statistica 5.0 PL (Statsoft Inc., Tulsa, OK, USA). The results were expres-sed as means and standard errors (± SEM). Comparisons between the experimental groups of ewes were performed using ANOVA and paired t-tests. Differences were consi-dered as significant at (p < 0.05).
The Local Animal Care and Use Committee approved all procedures used in this experiment.
Fig. 1. Experiment schedule Explanation: B. S. blood sampling
Ovulation rate and prolificacy. The data on repro-ductive performance in ewes treated with hCG (1 × hCG and 2 × hCG) as well as control groups (cont) have been presented in fig. 1. As has been shown in control ewes of both breeds, ovulation rate was higher than prolificacy. However, the differences in OR are
more accentuated in the PLS group whereas the proli-ficacy in BCP sheep. A more pronounced increase in OR was seen under the influ-ence of single pre-estral doses of hCG in the both breeds of ewes. Although the level of OR augmentation was more marked in PLS, the absolute value was higher in BCP ewes (2.6 and 2.9, respectively). The improve-ment in ovulation rate was not so remarkable as a result of the twofold administration of hCG. In this case the significant dif-ferences (p < 0.05) were found only in PLS ewes. In contrast to those changes, prolificacy rose considerably greater in ewes exposed to a double treat-ment with hCG, but significant differences were noted only in BCP ewes (fig. 2). We found that in all ewes used in this experiment the number of ovu-latory follicles rose together with the increment in the pla-sma IGF-1 level (tab. 1). The most influential level of IGF-1 on the number of ovulatory fol-licles following hCG treatment ranged between 28.0-31.0 ng/ml. IGF-I concentration. Plasma concentrations of IGF-1 tended (p < 0.01) to have greater con-centrations during the begin-ning of estrus in both control groups of ewes (PLS and BCP) (25.1 ng/ml and 26.3 ng/ml re-spectively) (fig. 3). In response to exposition on 1 × hCG signi-ficantly higher IGF-1concentra-tions were estimated (29.2 and Fig. 3. Influence of hCG treatment on plasma concentration of IGF-1 in two breeds of
ewes, +SEM
Explanation: as in fig. 2
Fig. 4. The plasma level of E2 and P4 in BCP ewes treated with hCG, +SEM Explanation: as in fig. 2
Fig. 2. Influence of hCG on ovulation rate (OR) and prolifi-cacy (Prol) in ywo breeds (PLS and BCP) of ewes, + SEM Explanation: * sig. difference compared with controls (P < 0.05) Tab. 1. Distribution of ovulatory follicles according to the
plasma level of IGF-1 (n = 30)
)l m / g n ( 1 -F G I Numberoffolilcles s e w e S L P BCPewes 0 , 2 2 -0 , 9 1 1 2 0 , 5 2 -0 , 2 2 1 3 0 , 8 2 -0 , 5 2 2 3 0 , 1 3 -0 , 8 2 3 4
30.1 ng/ml, respectively) at a time of estrus both in comparison to the control group and the group treated with 2 × hCG (26.5 and 27.9 ng/ml, respectively). The twofold treatment with hCG, with the exception of the response of BCP ewes after 24 hours from the first administration of hCG, only insignificantly changed the concentration of IGF-1. More responsive to hCG in these experimental conditions were the BCP sheep. E2 and P4 concentrations in BCP ewes. Before treatment with hCG plasma concentrations of E2 were similar in control and experimental ewes (fig. 4). Under the influence of hCG an elevated level of E2 occurred throughout the entire sampling period. BCP ewes treated with hCG had significantly (p < 0.05) greater E2 concentrations (1 × hCG: 20.4 pg/ml; 2 × hCG: 18.2 pg/ml) than ewes in the control group (16.1 pg/ml) on the day of expected ovulation. After 24 h from the second dose of hCG, the plasma of the E2 concentration dropped (17.2 pg/ml) in comparison to values noted in ewes subjected to only one dose of hCG (19.5 pg/ml).
After 24 h from the beginning of estrus there was a significant difference (p < 0.05) in P4 concentrations between control ewes (0.75 ng/ml) and those treated once (1.18 ng/ml) or twice (1.8 ng/ml) with hCG. The most stimulatory effect in reference to P4 on the day expected ovulation (48 hours after beginning open estrus) was seen after 2 × hCG treatment (1.95 ng/ml). In this case the concentration of P4 achieved the highest values amongst those observed in this experi-ment. As has been shown, the increasing level of P4 under the influence of hCG was dependent on the frequency of hCG treatment.
The contradictory results in respect to the improve-ment of the ovulation rate by treatimprove-ment with hCG (8, 10, 12, 13, 18-20, 25) incline us to distinguish withit in cyclic ewes of two breeds differing in respect to the number of ovulating follicles (PLS with typical single ovulation and BCP with prevalent double or even triple ovulation). In the present study, ovulation rate in response to single and twofold hCG administration around estrus time has been augmented. However, stimulatory effects of hCG were higher under the influence of a single dose of hCG, suggesting that it is more effective in the altering of follicular selection dynamics in the early follicular phase. The stimulatory response reported by us in respect to ovulation rate and prolificacy, especially seen in PLS ewes, found confirmation in the results given by McNatty et al. (15). They established that although the injected gonado-trophins, including LH, do not overcome the hierar-chical nature of follicular development during the fol-licular phase, they are able to increase oocyte yield. Because these authors also stressed that granulose cells of many follicles (> 2 mm diameter) did not share a similar cAMP response to such stimulation, this may be a reason for the discrepancy between the higher
stimulation of the ovulation rate than of prolificacy observed by us.
The stimulatory effect of hCG could be related to the increased plasma IGF-1 levels (21) detected in the estral BCP as well as PLS ewes. As it has been bril-liantly depicted by Driancourt el al. (5), the number and the range of hCG induced ovulations are similar to natural ovulation, but under the influence of hCG there is wide variation in OR with a higher percentage of low and high ovulations. Thus, these authors postu-lated that the systemic or local compounds are respon-sible for increasing the above mentioned variation. According to our results, in breeds such as PLS, hCG is able to induce an increase of IGF-1 production and secretion from additional follicles which subsequently achieve the ovulatory stage and in this way promotes OR. IGF-1 seems to improve the fertility by creating the beneficial growth factors milieu for selection and development of additional follicles that attain matura-tion and undergo ovulamatura-tion. In effect, hCG administra-tion augments steroidiogenesis seen as a rising plasma levels of estradiol, along with better a exhibition of estrus behavior. Such a response favors a better con-ception rate. Many authors emphasize that IGF-1 can be produced in the ovary by granulose cells, but espe-cially by fibroblasts and stromal cells (21, 23).
A higher level of IGF-1 found in the low prolific PLS breed is also in agreement with the data obtained by Spicer et. al. (22). According to the authors the plasma IGF-1 concentration is higher in breeds with a low ovulation rate. Thus, it seems that the augmented systemic IGF-1 level is responsible for the increased ovulation rate in some genotypes of ewes. Apart from IGFs, IGF binding proteins (IGFBPs) also unanalyzed by us have been implicated as potential regulators of follicular selection (22, 24). The last stage of follicle development, including additional follicles destined for ovulation, requires a decrease in IGFBPs, especially IGFBP2, with a simultaneous increase in IGF-1 pro-duction (21). Such a coordinated sequence of events is thought to provide support for facilitated follicular growth and may be the reason that the additional follicle achieves the ovulatory stage. As has been underlined by Webb et al. (26), when the number of ovulating follicles augments, fewer granullosa cells produce more estrogens and thus plasma E2 concen-tration is higher, as was in the case of our experiment. The obtained results showing the improvement in the ovulation rate under the influence of hCG treat-ment around the time of estrus are similar to those pre-sented by Khan et al. (12). But contrary to their data, we observed the augmentation of the P4 level in ewes treated twofold with hCG. The increased level of this gestagenic hormone under the influence of hCG may facilitate successive changes which proceed during the development of pregnancy. The augmented level of P4 under the influence of twofold hCG administration could find a response in higher prolificacy observed
ment given by Khan et al.(12), the augmentation of prolificacy following hCG treatment derived from the improvements in conceptus growth, placentation and embryo viability. When hCG was administrated twice, it could induce the development of accessory corpora lutea or improved corpus luteum functioning. It is also known that females with a higher level of P4 have more developed embryos.
Conclusions
In conclusion, the obtained results indicate that the optimal time to induce extra ovulatory response to hCG is the beginning of estrus, whereas hCG given at the end of estrus time is effective in improving prolificacy in ewes.
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Corresponding author: Prof. dr hab. Ryszard Bobowiec, Akademicka 12, 20-033 Lublin, Poland; e-mail: ryszard.bobowiec@up.lublin.pl
