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Superovulation in ewes by a single injection of pFSH dissolved in polyvinylpyrrolidone (PVP): effects of PVP molecular weight, concentration and schedule of treatment
Animal Reproduction Science 65 (2001) 255–264
Superovulation in ewes by a single injection of pFSH dissolved in polyvinylpyrrolidone (PVP): effects of PVP molecular weight, concentration and schedule of treatment A.G. D’Alessandro a,∗ , G. Martemucci a , M.A. Colonna b , A. Borghese c , M.G. Terzano c , A. Bellitti b a
Dipartimento PRO.GE.S.A., Università degli Studi di Bari, Via G. Amendola, 165/A, 70126 Bari, Italy Dipartimento S.A.V.A., Università degli Studi del Molise, Via De Sanctis, 86100 Campobasso, Italy c Istituto Sperimentale per la Zootecnia, Via Salaria, 131, Monterotondo, Rome, Italy
b
Received 17 August 2000; received in revised form 19 December 2000; accepted 19 December 2000
Abstract Three experiments were carried out to evaluate induction in ewes of superovulation and embryo production by a single injection of a porcine pituitary extract (pFSH) dissolved in polyvinylpyrrolidone (PVP), investigating the effects of PVP molecular weight and its concentration (Experiment I), time and method of treatment (Experiments II and III). All ewes were synchronized for estrus by vaginal sponges impregnated with fluorogestone acetate (FGA; 30 mg, 9 days) plus PGF2␣ (Cloprostenol, 50 g, 48 h before sponge removal — s.r.), and superovulated by 250 IU pFSH. In Experiment I, 60 Gentile di Puglia ewes were subdivided into five experimental groups (n = 12): Group A, the control, received six decreasing intramuscular (i.m.) doses of pFSH, 12 h apart, beginning 48 h before s.r.; Groups B and C were given 48 h before s.r. a single i.m. injection of pFSH dissolved in PVP with MW = 10,000, respectively, at concentrations of 15 and 30% w/v; Groups D and E received the same treatments as for B and C using PVP with MW = 40,000. None of the pFSH–PVP treatments were effective in inducing superovulation. In Experiment II, 22 Leccese ewes were subdivided into two groups (n = 11): Group A, control received i.m. four decreasing doses of pFSH, beginning 24 h before s.r., 12 h apart; Group B was given a single i.m. injection of pFSH dissolved in PVP (MW = 40,000 at 30% w/v), 24 h before s.r. The pFSH–PVP treatment provided an ovulation rate similar to the control and tended to enhance embryo yield (4.4 versus 2.4, P > 0.05). In Experiment III, 60 Leccese ewes were subdivided into six treatment groups (n = 10). Groups A and D served as controls and received i.m. 12 h apart, six doses (from 48 h before s.r.) ∗ Corresponding author. Tel.: +39-80-544-2825; fax: +39-80-544-2813. E-mail address: [email protected] (A.G. D’Alessandro).
0378-4320/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 1 ) 0 0 0 8 0 - X
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and four doses (from 24 h before s.r.) of pFSH, respectively. Groups B and C were treated by a single injection of pFSH in PVP (MW = 10,000; 30% w/v) 48 h before s.r., respectively by i.m. or subcutaneous (s.c.) administration. Groups E and F received the same treatments as for B and C 24 h before s.r. Intramuscular pFSH–PVP administration 24 h before s.r. provided an ovulation rate (8.1), mean numbers of ova recovered (5.6) and fertilized (4.2) comparable to the six or four dose treatments and significantly higher (P < 0.01) compared to the pFSH–PVP treatment carried out i.m. 48 h before s.r. These results show that a single injection of pFSH dissolved in PVP at 30% w/v, performed i.m. 24 h before s.r., is able to induce a superovulatory response comparable to that following multiple injection treatment, regardless of PVP molecular weight. © 2001 Elsevier Science B.V. All rights reserved. Keywords: pFSH; Polyvinylpyrrolidone; Ovarian response; Embryo production; Sheep-reproductive technology
1. Introduction Superovulation in sheep has been achieved by using several exogenous gonadotrophins, including porcine pituitary extracts which result in a satisfactory ovarian response and embryo production (Armstrong and Evans, 1983; Martemucci et al., 1988; Ryan et al., 1991). FSH is characterized by a short half-life (Akbar et al., 1974), therefore, it is administered by multiple doses injected twice daily for a 2–4 day period (Armstrong and Evans, 1983; Martemucci et al., 1988) in order to maintain bloodstream levels suitable to sustain growth of many follicles. Multiple injection regimen of superovulatory treatments can result stressful for animals, laborious, from the technical point of the view, and thus economically disadvantageous. Recent attempts have been made to simplify superovulatory protocols by reducing the number of doses of pFSH. Several studies conducted on cows (Takedomi et al., 1993; Yamamoto et al., 1993, 1994, 1995; Satoh et al., 1996; Sugano and Shinogi, 1999) have shown that superovulation may be induced by a single injection of FSH dissolved in polyvinylpyrrolidone (PVP). PVP is a synthetic organic polymer with different high molecular weights (10,000; 40,000 or 3,60,000), soluble in water and in a wide range of organic solvents. It is used for processing of so called “long acting” drugs, because of its properties to bind and to stabilize many molecules, thus prolonging their clearance rate in vivo. However, little is known about the factors which may affect PVP ability in acting as a vehicle for FSH. In cows, previous studies investigated the effect of PVP concentration in the solution used to dissolve FSH for the induction of superovulation (Takedomi et al., 1993) and it is assumed that the absorption of FSH from a subcutaneous (s.c.) deposit may occur more slowly than from an intramuscular (i.m.) one, due to the different extent of vascularization of the two sites (Bo et al., 1994). This study was undertaken to evaluate the effects of PVP molecular weight, its concentration and schedule of treatment with pFSH–PVP bolus, on the efficiency of PVP as a vehicle of pFSH administered in a single injection for superovulation and embryo production in ewes.
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2. Material and methods The study was carried out in Southern Italy (41◦ N latitude) on a total of 142 adult and dry ewes of two local breeds of sheep, Leccese and Gentile di Puglia, performing three experiments. In all the experiments, the estrus of ewes was synchronized by vaginal sponges impregnated with 30 mg fluorogestone acetate (FGA, Intervet, Milan, Italy) left in situ for 9 days. Forty-eight hours before sponge removal, ewes were injected i.m. with 50 g prostaglandin F2␣ (Cloprostenol, Estrumate, Schering-Plough). For the induction of superovulation, 250 International Units (IU) of a porcine pituitary extract (pFSH) having FSH/LH ratio of 1:1 was used (Pluset, Laboratorios Calier, Barcelona, Spain) (D’Alessandro et al., 1997). 2.1. Experiment I This experiment was designed to evaluate ovarian response following a single i.m. injection of pFSH dissolved in PVP in relation to PVP molecular weight (10,000 or 40,000) and its concentration (15 or 30% w/v) to vehicle pFSH. The trial was carried out in June on 60 Gentile di Puglia ewes. The synchronized ewes were subdivided into five homogenous groups (n = 12) corresponding to the experimental treatments. Group A — control, was treated with pFSH dissolved in saline following six i.m. decreasing doses (71.5, 71.5, 35.7, 35.7, 17.8, 17.8 IU), given 12 h apart, beginning from 48 h prior to sponge removal. The remaining four groups were treated by a single i.m. injection in the leg with pFSH dissolved in PVP, where PVP had molecular weight MW = 10,000 and concentration of 15% (Group B) or 30% (Group C), or MW = 40,000 and concentration of 15% (Group D) or 30% (Group E). For each molecular weight, two polyvinylpirrolidone solutions were prepared in order to dissolve pFSH. For the 30% solution, 37.5 g of PVP 10,000 (PVP-10, Sigma, St. Louis, USA) or PVP 40,000 (PVP-40, Sigma) were dissolved with distilled water up to a final volume of 100 mL; for the 15% solution, 18.75% (w/v) PVP solutions of each molecular weight were prepared. All the solutions were sterilized by autoclaving and stored at 4◦ C until use. Afterwards, the total dose of pFSH administered per ewe (250 IU) was first dissolved in 1 mL of saline and then mixed to 4 ml of either the 37.5 or 18.75% PVP solutions. Ewes were checked for estrus beginning 16 h after sponge removal, every 4 h, with the aid of teaser rams. On the 7th day after sponge removal, ewes underwent laparoscopy (Martemucci et al., 1984) in order to evaluate ovarian response. Ovaries were examined and the number of corpora lutea either normal (>3 mm) and anomalous (≤3 mm) and large unovulated follicles (>4 mm) were recorded. Ewes showing more than three corpora lutea were considered as superovulated (D’Alessandro et al., 1997). 2.2. Experiment II This experiment was drawn up on the basis of the preliminary information gained from Experiment I. It was performed in order to investigate the efficiency of pFSH in PVP with MW = 40,000 at a concentration of 30%, administered 24 h before sponge removal, on induction of superovulation and embryo production.
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Twenty-two Leccese sheep were treated to induce estrus in March–April, corresponding to seasonal anoestrus (Martemucci et al., 1980), and randomly subdivided into two treatment groups. Group (A) — control (n = 11) was given pFSH dissolved in saline and administered in four decreasing doses (100, 75, 50, 25 IU), beginning 24 h before sponge removal; Group (B) (n = 11) — received a single i.m. injection of pFSH dissolved in PVP (MW = 40,000; final concentration 30%) 24 h before pessary removal. 2.3. Experiment III This experiment was designed to gain further insight on pFSH–PVP treatment in a single injection in relation to the effects of administration time (48 or 24 h before pessary removal) and method of pFSH–PVP administration (i.m. or s.c.), on superovulatory response. For this experiment we used PVP having MW = 10,000 and final concentration of 30% (w/v) to dissolve pFSH. In October–November, corresponding to the breeding season (Martemucci et al., 1980), a total of 60 Leccese ewes were randomly divided into six experimental treatment groups (n = 10). Of these groups, two served as controls and received pFSH dissolved in saline by multiple i.m. injections in the leg: Group A was treated with six decreasing doses (71.5, 71.5, 35.7, 35.7, 17.8, 17.8 IU) over 3 days starting 48 h before sponge withdrawal; Groups D received four doses (100, 75, 50, 25 IU) in 2 days, beginning 24 h before sponge removal. The remaining four groups were given a single injection of pFSH dissolved in PVP administered: at 48 h before sponge removal by i.m. injection, Group B or s.c. injection, Group C, or at 24 h before sponge removal i.m., Group E or s.c. Group F. Intramuscular pFSH–PVP injection was performed in the leg (Groups B and E) while s.c. injection behind the shoulder was given to groups C and F. Ewes considered in Experiment II and III were checked for estrus following the same method as in Experiment I. At the beginning of sexual receptivity ewes were hand-mated every 6–8 h onwards until standing estrus. On the 7th day after sponge removal, ewes were laparotomized under general anesthesia to estimate ovarian response (numbers of corpora lutea and follicles >4 mm) and to collect ova, using the procedure previously described (Martemucci et al., 1988). Ova recovery was accomplished by the retrograde flushing of each uterine horn with 20 mL Whittingham’s modified PBS (Whittingham, 1971) supplemented with 10% fetal calf serum (FCS). The flushing medium was observed under a stereomicroscope (40× magnification) then the ova were examined under a phase contrast microscope (225×), with cleavage taken as evidence for fertilization. According to their stage of development in relation to estrus and to their morphological features, embryos were graded as excellent, good and fair, considering as transferable only the first two classes (Martemucci et al., 1988). 2.4. Statistical analysis Data referring to the onset of estrus and to the numbers of corpora lutea, follicles >4 mm, ova collected, ova fertilized and transferable embryos were analysed by least square analysis of variance using the GLM procedure of the SAS (SAS, 1987).
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Table 1 Experiment I, ovarian response in Gentile di Puglia ewes treated with pFSH (250 IU) administered by multiple injections or by a single i.m. dose of pFSH dissolved in PVP with different molecular weights (MW, 10,000 or 40,000) and concentration (15 or 30%, w/v), given 48 h before sponge removal (mean ± S.E.M.; percentages in brackets)a Treatment
No. of ewes Treated In estrus Ovulated Superovulated Interval pessary removal, onset of estrus (h) Ovulation rate CL ≤ 3 mm Follicles > 4 mm a b
pFSH in six dosesb , control (Group A)
pFSH in a Single dose dissolved in PVP MW = 10,000
MW = 40,000
PVP 15% (Group B)
PVP 30% (Group C)
PVP 15% (Group D)
PVP 30% (Group E)
12 12 11 10
12 8 11 0
12 9 11 0
12 11 10 0
12 11 12 3
27.3 ± 5.5 B
50.7 ± 6.8 A
35.5 ± 6.4
50.5 ± 5.8 A
39.4 ± 5.8
10.2 ± 1.0 A 11.9 0.9 ± 0.5
0.9 ± 0.9 B 40.9 a 0.2 ± 0.5 b
1.4 ± 1.0 B 13.6 1.8 ± 0.5 a
1.0 ± 1.0 B 31.7 1.4 ± 0.5
3.0 ± 1.0 B 8.3 b 0.8 ± 0.5
Values with different letters in the row differ significantly: A, B (P < 0.01); a, b (P < 0.05). The pFSH doses dissolved in saline and injected twice daily, 12 h apart, beginning 48 h before s.r.
Data were analysed considering the fixed effect of treatment in the statistical model. Student’s t-test with the predicted difference (PDIFF) option of the GLM was used to compare least square means while Chi-square test was used for the percentage values (SAS, 1987).
3. Results 3.1. Experiment I Hormone treatment following a single injection of pFSH dissolved in PVP was not effective in inducing superovulation (Table 1). Of the four groups treated by pFSH–PVP single injection, only in Group E, corresponding to PVP with MW = 40,000 and concentration of 30% (w/v), there was induction of superovulation, estimated as percentage of ewes having equal to or more than three corpora lutea, in 40% of treated ewes. In the remaining groups (B-C-D) no ewes were superovulated. Treatment by pFSH–PVP single injection delayed the exhibition of estrus compared to the multiple injection pFSH treatment (Group A; P < 0.05). In particular, the delay was significant (P < 0.01) when PVP was used at a lower concentration (15% w/v) for both the PVP molecular weights (10,000 and 40,000; Groups B, and D). The ovulation rate was markedly (P < 0.01) lowered in all groups treated by a single administration of pFSH in PVP compared to the control (10.2 corpora lutea). Within the pFSH–PVP treatments, administration of pFSH in a 30% vehicle of PVP 40,000 (Group E) provided a slightly better ovulation rate (3.0 versus 0.9–1.4; P > 0.05).
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Table 2 Experiment II, ovarian response and embryo production in Leccese ewes treated with pFSH (250 IU) administered by multiple injections or by a single i.m. dose of pFSH dissolved in PVP (MW = 40,000; 30% w/v solution) given 24 h before sponge removal (mean ± S.E.M.)a Treatment No. of ewes Treated In estrus and ovulated Superovulated Interval pessary removal– onset of estrus (h) Ovulation rate Follicles > 4 mm Ova recovered Ova fertilized Transferable embryos (%) a b
pFSH in four dosesb , control (Group A) 11 11 9
pFSH in a Single dose dissolved in PVP (MW = 40,000; 30%) (Group B) 8 8 7
27.9 ± 2.6 A
40.2 ± 3.0 B
6.6 ± 1.0 0.4 ± 0.2 b 4.0 ± 1.0 2.4 ± 0.9 83.3
6.2 ± 1.2 1.1 ± 0.2 a 5.6 ± 1.2 4.4 ± 1.0 75.0
Values with different letters in the row differ significantly: A, B (P < 0.01); a, b (P < 0.05). The pFSH doses dissolved in saline and injected twice daily, 12 h apart, beginning 24 h before s.r.
3.2. Experiment II Three ewes assigned to Group B were excluded from statistical data processing due to the presence of ovarian adhesions (Table 2). The percentage of superovulated ewes was similar in treatments A and B (82.0 and 87.0%; P > 0.05). Also in this trial, the single administration of pFSH in PVP delayed the onset of estrus compared to the control (P < 0.01) while the ovulation rate was similar in the two treatment groups (6.2 and 6.6, respectively). Treatment B increased the mean number of unovulated follicles (P < 0.05) (Table 2). Compared to pFSH control treatment, pFSH–PVP administration showed a trend (P > 0.05) towards an increase of mean numbers of recovered ova, fertilization rate (82.4% versus 57.4%) and embryo yield (4.4 versus 2.4). There were no differences in the quality of embryos producted (percentage of transferable embryos). 3.3. Experiment III Two ewes from Group F were excluded from statistical analysis due to pessary loss. Among the treatments performed 48 h before sponge removal, the single pFSH–PVP i.m. injection induced a superovulatory response only in 10% of ewes compared to 50% of s.c. pFSH–PVP treatment or 60% of the control (Table 3). In both pFSH–PVP treated groups (B and C), onset of estrus was delayed (P < 0.01) and the ovulation rate resulted significantly lower compared to the control, mainly when performed by i.m. injection (P < 0.01). Superovulatory treatment by six pFSH injections (Group A) significantly advanced estrus (P < 0.01) and increased ovulation rate (0.05 > P < 0.01) compared to the pFSH–PVP treatments (Groups B and C) (Table 3).Within the 24 h before sponge removal treatment, no differences were found among the treatments. However, the s.c. pFSH–PVP
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administration method affected the lowest proportion of superovulated ewes (20% versus 60–70% of Groups E and D), while i.m. pFSH–PVP treatment tended to increase ovulation rate (8.1) compared to the control (5.9, Group D; P > 0.05) and resulted similar to the six pFSH dose treatment (8.9, Group A). Considering i.m. injection of pFSH–PVP treatment, the influence of time of administration was significant resulting in more advanced onset of estrus (P < 0.01), higher induction of superovulation and ovulatory response (P < 0.01) in ewes treated 24 h before sponge removal. On the contrary, the pFSH–PVP treatments injected subcutaneously differed only for the onset of estrus, which occurred earlier following administration 24 h before sponge removal (Group F versus Group C; P < 0.01). Compared to Group A, both the pFSH–PVP treatments performed 48 h before sponge removal, as a single injection either i.m. or s.c., decreased ova recovery (0.05 > P < 0.01) and embryo production (0.8–1.9 for Groups B and C, versus 4.6 embryos of Group A). On the contrary, i.m. administration of a single pFSH–PVP bolus 24 h (Group E) rather than 48 h before sponge removal (Group B) increased the mean number of ova recovered (5.6 versus 1.0; P < 0.01) and tended (P > 0.05) to improve the average of embryos yielded (4.2 versus 0.8) as well as the proportion of good quality embryos (81.0% versus 66.7%). No differences were shown between Group E and the control Groups D and A.
4. Discussion In Experiment I, treatment with pFSH dissolved in PVP and administered by a single injection was not effective in inducing superovulation in Gentile di Puglia ewes. We hypothesized that the ineffectiveness of this treatment was likely to be attributable to the inadequate administration time of pFSH–PVP bolus, which was at the 48th hour before the end of progestagen treatment. However, from this experiment some indications could be obtained concerning the influences of PVP molecular weight and its concentration in solution to dissolve pFSH. The use of PVP with higher molecular weight (40,000 versus 10,000) resulted only in a slight trend toward an increase of ovulatory response, leading us to hypothesize that the different molecular weight of the two PVP polymers does not influence PVP properties in prolonging pFSH half-life. Adversely, efficiency of the treatment performed by pFSH dissolved in PVP and administered in a single injection, seems to be influenced by the concentration of PVP in the solution. In fact, better results corresponded to the higher PVP rate (30% versus 15%), mainly as far as the time of estrus onset (more advanced), and the lower incidence of anomalous corpora lutea. It is possible that 30% rather than 15% PVP in the solution stabilizes pFSH molecules better and this could positively affect the events involved in the occurence of estrus and the luteinizing processes of corpora lutea. In cows, a higher ovulation rate and embryo production was reported when pFSH was dissolved in a higher solution of PVP (50%) having lower molecular weight (40,000) rather than lower solution of PVP (25%) but with higher molecular weight (360,000) (Takedomi et al., 1995). In Experiment II, we wanted to test the efficiency of the pFSH–PVP treatment in a single i.m. injection, changing the administration time of the hormonal preparation in PVP with MW = 40,000, from 48 (Experiment I) to 24 h before the end of progestagen treatment. In effect, this time resulted effective in the induction of superovulation and provided an embryo production slightly higher compared to the control. These results were confirmed
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in Experiment III, where we used PVP having MW = 10,000. In this trial, the importance of treatment time but also the method of administration of pFSH–PVP bolus was emphasized. Injection of pFSH dissolved in PVP 48 h before pessary withdrawal induced a significant delay of estrus onset, a low ovulation rate and a poor embryo production, following either i.m. or s.c. administration. The administration method of pFSH–PVP plays a role in the efficiency of superovulatory treatment. It was reported (Kelly et al., 1997) that pFSH was released into the bloodstream more slowly from a s.c. deposit than from an i.m. site, maintaining higher circulating levels of gonadotrophin over a longer time. Moreover, FSH absorbtion rate is affected by the amount of fat present at the site of injection, as referred by Bo et al. (1994) in a study on cows. The low superovulatory response obtained in ewes treated with pFSH–PVP bolus 48 h before sponge removal could be ascribable to the high circulating levels of the gonadotrophin, due to the administration of 250 IU pFSH all at once, in advance compared to the physiological events regulating ovulation rate. It was demonstrated that in sheep recruitment of follicles occurs after the luteolysis (Driancourt et al., 1985). In our study, administration of the gonadotrophin coincided with the induction of luteolysis by PGF2␣ injection (at 48th hour before sponge removal). Thus, we can deduce that at this time PVP was unable to maintain sufficient pFSH activity to sustain growth of multiple follicles, even if following s.c. administration.The best superovulatory response was obtained following pFSH–PVP treatment 24 h before sponge removal, but performing the single hormone administration by i.m. injection, confirming the findings reported by Dattena et al. (1994) in Sarda ewes. Ewe genotype may also play an important role in ovarian response to the hormone treatment as unsatisfactory results were reported in Romanov ewes (Lajous et al., 1997) as opposed to the positive results in Sarda ewes (Dattena et al., 1994). In conclusion, the results of this study demonstrate the efficiency for superovulation of PVP having molecular weight of 40,000 or 10,000 at 30% w/v as vehicle for treatment with 250 IU pFSH in a single administration, when performed at the 24th hour before the end of progestagen treatment by i.m. injection.
Acknowledgements This research was supported by the Ministero delle Politiche Agricole e Forestali, Special Project RAIZ, Publication No. RZ 257. The authors wish to thank L. Bongermino for statistical processing analysis and L. Basso for technical assistance. References Akbar, A.M., Nett, T.M., Niswender, G.D., 1974. Metabolic clearance and secretion rates of gonadotropins at different stages of the estrous cycle in ewes. Endocrinology 94, 1318–1324. Armstrong, D.T., Evans, G., 1983. Factors influencing success of embryo tansfer in sheep and goats. Theriogenology 39, 7–24. Bo, G.A., Hockley, D.K., Nasser, L.F., Mapletoft, R.J., 1994. Superovulatory response to a single subcutaneous injection of Folltropin in beef cattle. Theriogenology 42, 963–975. D’Alessandro, A., Martemucci, G., Colonna, M.A., Cafueri, C., Toteda, F., 1997. Some effects of adding p-LH in defined amounts to purified pFSH to modify FSH/LH ratios during the superovulatory treatment of anoestrus ewes. Anim. Reprod. Sci. 47, 91–98.
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Superovulation in ewes by a single injection of pFSH dissolved in polyvinylpyrrolidone (PVP): effects of PVP molecular weight, concentration and schedule of treatment A.G. D’Alessandro a,∗ , G. Martemucci a , M.A. Colonna b , A. Borghese c , M.G. Terzano c , A. Bellitti b a
Dipartimento PRO.GE.S.A., Università degli Studi di Bari, Via G. Amendola, 165/A, 70126 Bari, Italy Dipartimento S.A.V.A., Università degli Studi del Molise, Via De Sanctis, 86100 Campobasso, Italy c Istituto Sperimentale per la Zootecnia, Via Salaria, 131, Monterotondo, Rome, Italy
b
Received 17 August 2000; received in revised form 19 December 2000; accepted 19 December 2000
Abstract Three experiments were carried out to evaluate induction in ewes of superovulation and embryo production by a single injection of a porcine pituitary extract (pFSH) dissolved in polyvinylpyrrolidone (PVP), investigating the effects of PVP molecular weight and its concentration (Experiment I), time and method of treatment (Experiments II and III). All ewes were synchronized for estrus by vaginal sponges impregnated with fluorogestone acetate (FGA; 30 mg, 9 days) plus PGF2␣ (Cloprostenol, 50 g, 48 h before sponge removal — s.r.), and superovulated by 250 IU pFSH. In Experiment I, 60 Gentile di Puglia ewes were subdivided into five experimental groups (n = 12): Group A, the control, received six decreasing intramuscular (i.m.) doses of pFSH, 12 h apart, beginning 48 h before s.r.; Groups B and C were given 48 h before s.r. a single i.m. injection of pFSH dissolved in PVP with MW = 10,000, respectively, at concentrations of 15 and 30% w/v; Groups D and E received the same treatments as for B and C using PVP with MW = 40,000. None of the pFSH–PVP treatments were effective in inducing superovulation. In Experiment II, 22 Leccese ewes were subdivided into two groups (n = 11): Group A, control received i.m. four decreasing doses of pFSH, beginning 24 h before s.r., 12 h apart; Group B was given a single i.m. injection of pFSH dissolved in PVP (MW = 40,000 at 30% w/v), 24 h before s.r. The pFSH–PVP treatment provided an ovulation rate similar to the control and tended to enhance embryo yield (4.4 versus 2.4, P > 0.05). In Experiment III, 60 Leccese ewes were subdivided into six treatment groups (n = 10). Groups A and D served as controls and received i.m. 12 h apart, six doses (from 48 h before s.r.) ∗ Corresponding author. Tel.: +39-80-544-2825; fax: +39-80-544-2813. E-mail address: [email protected] (A.G. D’Alessandro).
0378-4320/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 1 ) 0 0 0 8 0 - X
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and four doses (from 24 h before s.r.) of pFSH, respectively. Groups B and C were treated by a single injection of pFSH in PVP (MW = 10,000; 30% w/v) 48 h before s.r., respectively by i.m. or subcutaneous (s.c.) administration. Groups E and F received the same treatments as for B and C 24 h before s.r. Intramuscular pFSH–PVP administration 24 h before s.r. provided an ovulation rate (8.1), mean numbers of ova recovered (5.6) and fertilized (4.2) comparable to the six or four dose treatments and significantly higher (P < 0.01) compared to the pFSH–PVP treatment carried out i.m. 48 h before s.r. These results show that a single injection of pFSH dissolved in PVP at 30% w/v, performed i.m. 24 h before s.r., is able to induce a superovulatory response comparable to that following multiple injection treatment, regardless of PVP molecular weight. © 2001 Elsevier Science B.V. All rights reserved. Keywords: pFSH; Polyvinylpyrrolidone; Ovarian response; Embryo production; Sheep-reproductive technology
1. Introduction Superovulation in sheep has been achieved by using several exogenous gonadotrophins, including porcine pituitary extracts which result in a satisfactory ovarian response and embryo production (Armstrong and Evans, 1983; Martemucci et al., 1988; Ryan et al., 1991). FSH is characterized by a short half-life (Akbar et al., 1974), therefore, it is administered by multiple doses injected twice daily for a 2–4 day period (Armstrong and Evans, 1983; Martemucci et al., 1988) in order to maintain bloodstream levels suitable to sustain growth of many follicles. Multiple injection regimen of superovulatory treatments can result stressful for animals, laborious, from the technical point of the view, and thus economically disadvantageous. Recent attempts have been made to simplify superovulatory protocols by reducing the number of doses of pFSH. Several studies conducted on cows (Takedomi et al., 1993; Yamamoto et al., 1993, 1994, 1995; Satoh et al., 1996; Sugano and Shinogi, 1999) have shown that superovulation may be induced by a single injection of FSH dissolved in polyvinylpyrrolidone (PVP). PVP is a synthetic organic polymer with different high molecular weights (10,000; 40,000 or 3,60,000), soluble in water and in a wide range of organic solvents. It is used for processing of so called “long acting” drugs, because of its properties to bind and to stabilize many molecules, thus prolonging their clearance rate in vivo. However, little is known about the factors which may affect PVP ability in acting as a vehicle for FSH. In cows, previous studies investigated the effect of PVP concentration in the solution used to dissolve FSH for the induction of superovulation (Takedomi et al., 1993) and it is assumed that the absorption of FSH from a subcutaneous (s.c.) deposit may occur more slowly than from an intramuscular (i.m.) one, due to the different extent of vascularization of the two sites (Bo et al., 1994). This study was undertaken to evaluate the effects of PVP molecular weight, its concentration and schedule of treatment with pFSH–PVP bolus, on the efficiency of PVP as a vehicle of pFSH administered in a single injection for superovulation and embryo production in ewes.
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2. Material and methods The study was carried out in Southern Italy (41◦ N latitude) on a total of 142 adult and dry ewes of two local breeds of sheep, Leccese and Gentile di Puglia, performing three experiments. In all the experiments, the estrus of ewes was synchronized by vaginal sponges impregnated with 30 mg fluorogestone acetate (FGA, Intervet, Milan, Italy) left in situ for 9 days. Forty-eight hours before sponge removal, ewes were injected i.m. with 50 g prostaglandin F2␣ (Cloprostenol, Estrumate, Schering-Plough). For the induction of superovulation, 250 International Units (IU) of a porcine pituitary extract (pFSH) having FSH/LH ratio of 1:1 was used (Pluset, Laboratorios Calier, Barcelona, Spain) (D’Alessandro et al., 1997). 2.1. Experiment I This experiment was designed to evaluate ovarian response following a single i.m. injection of pFSH dissolved in PVP in relation to PVP molecular weight (10,000 or 40,000) and its concentration (15 or 30% w/v) to vehicle pFSH. The trial was carried out in June on 60 Gentile di Puglia ewes. The synchronized ewes were subdivided into five homogenous groups (n = 12) corresponding to the experimental treatments. Group A — control, was treated with pFSH dissolved in saline following six i.m. decreasing doses (71.5, 71.5, 35.7, 35.7, 17.8, 17.8 IU), given 12 h apart, beginning from 48 h prior to sponge removal. The remaining four groups were treated by a single i.m. injection in the leg with pFSH dissolved in PVP, where PVP had molecular weight MW = 10,000 and concentration of 15% (Group B) or 30% (Group C), or MW = 40,000 and concentration of 15% (Group D) or 30% (Group E). For each molecular weight, two polyvinylpirrolidone solutions were prepared in order to dissolve pFSH. For the 30% solution, 37.5 g of PVP 10,000 (PVP-10, Sigma, St. Louis, USA) or PVP 40,000 (PVP-40, Sigma) were dissolved with distilled water up to a final volume of 100 mL; for the 15% solution, 18.75% (w/v) PVP solutions of each molecular weight were prepared. All the solutions were sterilized by autoclaving and stored at 4◦ C until use. Afterwards, the total dose of pFSH administered per ewe (250 IU) was first dissolved in 1 mL of saline and then mixed to 4 ml of either the 37.5 or 18.75% PVP solutions. Ewes were checked for estrus beginning 16 h after sponge removal, every 4 h, with the aid of teaser rams. On the 7th day after sponge removal, ewes underwent laparoscopy (Martemucci et al., 1984) in order to evaluate ovarian response. Ovaries were examined and the number of corpora lutea either normal (>3 mm) and anomalous (≤3 mm) and large unovulated follicles (>4 mm) were recorded. Ewes showing more than three corpora lutea were considered as superovulated (D’Alessandro et al., 1997). 2.2. Experiment II This experiment was drawn up on the basis of the preliminary information gained from Experiment I. It was performed in order to investigate the efficiency of pFSH in PVP with MW = 40,000 at a concentration of 30%, administered 24 h before sponge removal, on induction of superovulation and embryo production.
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Twenty-two Leccese sheep were treated to induce estrus in March–April, corresponding to seasonal anoestrus (Martemucci et al., 1980), and randomly subdivided into two treatment groups. Group (A) — control (n = 11) was given pFSH dissolved in saline and administered in four decreasing doses (100, 75, 50, 25 IU), beginning 24 h before sponge removal; Group (B) (n = 11) — received a single i.m. injection of pFSH dissolved in PVP (MW = 40,000; final concentration 30%) 24 h before pessary removal. 2.3. Experiment III This experiment was designed to gain further insight on pFSH–PVP treatment in a single injection in relation to the effects of administration time (48 or 24 h before pessary removal) and method of pFSH–PVP administration (i.m. or s.c.), on superovulatory response. For this experiment we used PVP having MW = 10,000 and final concentration of 30% (w/v) to dissolve pFSH. In October–November, corresponding to the breeding season (Martemucci et al., 1980), a total of 60 Leccese ewes were randomly divided into six experimental treatment groups (n = 10). Of these groups, two served as controls and received pFSH dissolved in saline by multiple i.m. injections in the leg: Group A was treated with six decreasing doses (71.5, 71.5, 35.7, 35.7, 17.8, 17.8 IU) over 3 days starting 48 h before sponge withdrawal; Groups D received four doses (100, 75, 50, 25 IU) in 2 days, beginning 24 h before sponge removal. The remaining four groups were given a single injection of pFSH dissolved in PVP administered: at 48 h before sponge removal by i.m. injection, Group B or s.c. injection, Group C, or at 24 h before sponge removal i.m., Group E or s.c. Group F. Intramuscular pFSH–PVP injection was performed in the leg (Groups B and E) while s.c. injection behind the shoulder was given to groups C and F. Ewes considered in Experiment II and III were checked for estrus following the same method as in Experiment I. At the beginning of sexual receptivity ewes were hand-mated every 6–8 h onwards until standing estrus. On the 7th day after sponge removal, ewes were laparotomized under general anesthesia to estimate ovarian response (numbers of corpora lutea and follicles >4 mm) and to collect ova, using the procedure previously described (Martemucci et al., 1988). Ova recovery was accomplished by the retrograde flushing of each uterine horn with 20 mL Whittingham’s modified PBS (Whittingham, 1971) supplemented with 10% fetal calf serum (FCS). The flushing medium was observed under a stereomicroscope (40× magnification) then the ova were examined under a phase contrast microscope (225×), with cleavage taken as evidence for fertilization. According to their stage of development in relation to estrus and to their morphological features, embryos were graded as excellent, good and fair, considering as transferable only the first two classes (Martemucci et al., 1988). 2.4. Statistical analysis Data referring to the onset of estrus and to the numbers of corpora lutea, follicles >4 mm, ova collected, ova fertilized and transferable embryos were analysed by least square analysis of variance using the GLM procedure of the SAS (SAS, 1987).
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Table 1 Experiment I, ovarian response in Gentile di Puglia ewes treated with pFSH (250 IU) administered by multiple injections or by a single i.m. dose of pFSH dissolved in PVP with different molecular weights (MW, 10,000 or 40,000) and concentration (15 or 30%, w/v), given 48 h before sponge removal (mean ± S.E.M.; percentages in brackets)a Treatment
No. of ewes Treated In estrus Ovulated Superovulated Interval pessary removal, onset of estrus (h) Ovulation rate CL ≤ 3 mm Follicles > 4 mm a b
pFSH in six dosesb , control (Group A)
pFSH in a Single dose dissolved in PVP MW = 10,000
MW = 40,000
PVP 15% (Group B)
PVP 30% (Group C)
PVP 15% (Group D)
PVP 30% (Group E)
12 12 11 10
12 8 11 0
12 9 11 0
12 11 10 0
12 11 12 3
27.3 ± 5.5 B
50.7 ± 6.8 A
35.5 ± 6.4
50.5 ± 5.8 A
39.4 ± 5.8
10.2 ± 1.0 A 11.9 0.9 ± 0.5
0.9 ± 0.9 B 40.9 a 0.2 ± 0.5 b
1.4 ± 1.0 B 13.6 1.8 ± 0.5 a
1.0 ± 1.0 B 31.7 1.4 ± 0.5
3.0 ± 1.0 B 8.3 b 0.8 ± 0.5
Values with different letters in the row differ significantly: A, B (P < 0.01); a, b (P < 0.05). The pFSH doses dissolved in saline and injected twice daily, 12 h apart, beginning 48 h before s.r.
Data were analysed considering the fixed effect of treatment in the statistical model. Student’s t-test with the predicted difference (PDIFF) option of the GLM was used to compare least square means while Chi-square test was used for the percentage values (SAS, 1987).
3. Results 3.1. Experiment I Hormone treatment following a single injection of pFSH dissolved in PVP was not effective in inducing superovulation (Table 1). Of the four groups treated by pFSH–PVP single injection, only in Group E, corresponding to PVP with MW = 40,000 and concentration of 30% (w/v), there was induction of superovulation, estimated as percentage of ewes having equal to or more than three corpora lutea, in 40% of treated ewes. In the remaining groups (B-C-D) no ewes were superovulated. Treatment by pFSH–PVP single injection delayed the exhibition of estrus compared to the multiple injection pFSH treatment (Group A; P < 0.05). In particular, the delay was significant (P < 0.01) when PVP was used at a lower concentration (15% w/v) for both the PVP molecular weights (10,000 and 40,000; Groups B, and D). The ovulation rate was markedly (P < 0.01) lowered in all groups treated by a single administration of pFSH in PVP compared to the control (10.2 corpora lutea). Within the pFSH–PVP treatments, administration of pFSH in a 30% vehicle of PVP 40,000 (Group E) provided a slightly better ovulation rate (3.0 versus 0.9–1.4; P > 0.05).
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Table 2 Experiment II, ovarian response and embryo production in Leccese ewes treated with pFSH (250 IU) administered by multiple injections or by a single i.m. dose of pFSH dissolved in PVP (MW = 40,000; 30% w/v solution) given 24 h before sponge removal (mean ± S.E.M.)a Treatment No. of ewes Treated In estrus and ovulated Superovulated Interval pessary removal– onset of estrus (h) Ovulation rate Follicles > 4 mm Ova recovered Ova fertilized Transferable embryos (%) a b
pFSH in four dosesb , control (Group A) 11 11 9
pFSH in a Single dose dissolved in PVP (MW = 40,000; 30%) (Group B) 8 8 7
27.9 ± 2.6 A
40.2 ± 3.0 B
6.6 ± 1.0 0.4 ± 0.2 b 4.0 ± 1.0 2.4 ± 0.9 83.3
6.2 ± 1.2 1.1 ± 0.2 a 5.6 ± 1.2 4.4 ± 1.0 75.0
Values with different letters in the row differ significantly: A, B (P < 0.01); a, b (P < 0.05). The pFSH doses dissolved in saline and injected twice daily, 12 h apart, beginning 24 h before s.r.
3.2. Experiment II Three ewes assigned to Group B were excluded from statistical data processing due to the presence of ovarian adhesions (Table 2). The percentage of superovulated ewes was similar in treatments A and B (82.0 and 87.0%; P > 0.05). Also in this trial, the single administration of pFSH in PVP delayed the onset of estrus compared to the control (P < 0.01) while the ovulation rate was similar in the two treatment groups (6.2 and 6.6, respectively). Treatment B increased the mean number of unovulated follicles (P < 0.05) (Table 2). Compared to pFSH control treatment, pFSH–PVP administration showed a trend (P > 0.05) towards an increase of mean numbers of recovered ova, fertilization rate (82.4% versus 57.4%) and embryo yield (4.4 versus 2.4). There were no differences in the quality of embryos producted (percentage of transferable embryos). 3.3. Experiment III Two ewes from Group F were excluded from statistical analysis due to pessary loss. Among the treatments performed 48 h before sponge removal, the single pFSH–PVP i.m. injection induced a superovulatory response only in 10% of ewes compared to 50% of s.c. pFSH–PVP treatment or 60% of the control (Table 3). In both pFSH–PVP treated groups (B and C), onset of estrus was delayed (P < 0.01) and the ovulation rate resulted significantly lower compared to the control, mainly when performed by i.m. injection (P < 0.01). Superovulatory treatment by six pFSH injections (Group A) significantly advanced estrus (P < 0.01) and increased ovulation rate (0.05 > P < 0.01) compared to the pFSH–PVP treatments (Groups B and C) (Table 3).Within the 24 h before sponge removal treatment, no differences were found among the treatments. However, the s.c. pFSH–PVP
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administration method affected the lowest proportion of superovulated ewes (20% versus 60–70% of Groups E and D), while i.m. pFSH–PVP treatment tended to increase ovulation rate (8.1) compared to the control (5.9, Group D; P > 0.05) and resulted similar to the six pFSH dose treatment (8.9, Group A). Considering i.m. injection of pFSH–PVP treatment, the influence of time of administration was significant resulting in more advanced onset of estrus (P < 0.01), higher induction of superovulation and ovulatory response (P < 0.01) in ewes treated 24 h before sponge removal. On the contrary, the pFSH–PVP treatments injected subcutaneously differed only for the onset of estrus, which occurred earlier following administration 24 h before sponge removal (Group F versus Group C; P < 0.01). Compared to Group A, both the pFSH–PVP treatments performed 48 h before sponge removal, as a single injection either i.m. or s.c., decreased ova recovery (0.05 > P < 0.01) and embryo production (0.8–1.9 for Groups B and C, versus 4.6 embryos of Group A). On the contrary, i.m. administration of a single pFSH–PVP bolus 24 h (Group E) rather than 48 h before sponge removal (Group B) increased the mean number of ova recovered (5.6 versus 1.0; P < 0.01) and tended (P > 0.05) to improve the average of embryos yielded (4.2 versus 0.8) as well as the proportion of good quality embryos (81.0% versus 66.7%). No differences were shown between Group E and the control Groups D and A.
4. Discussion In Experiment I, treatment with pFSH dissolved in PVP and administered by a single injection was not effective in inducing superovulation in Gentile di Puglia ewes. We hypothesized that the ineffectiveness of this treatment was likely to be attributable to the inadequate administration time of pFSH–PVP bolus, which was at the 48th hour before the end of progestagen treatment. However, from this experiment some indications could be obtained concerning the influences of PVP molecular weight and its concentration in solution to dissolve pFSH. The use of PVP with higher molecular weight (40,000 versus 10,000) resulted only in a slight trend toward an increase of ovulatory response, leading us to hypothesize that the different molecular weight of the two PVP polymers does not influence PVP properties in prolonging pFSH half-life. Adversely, efficiency of the treatment performed by pFSH dissolved in PVP and administered in a single injection, seems to be influenced by the concentration of PVP in the solution. In fact, better results corresponded to the higher PVP rate (30% versus 15%), mainly as far as the time of estrus onset (more advanced), and the lower incidence of anomalous corpora lutea. It is possible that 30% rather than 15% PVP in the solution stabilizes pFSH molecules better and this could positively affect the events involved in the occurence of estrus and the luteinizing processes of corpora lutea. In cows, a higher ovulation rate and embryo production was reported when pFSH was dissolved in a higher solution of PVP (50%) having lower molecular weight (40,000) rather than lower solution of PVP (25%) but with higher molecular weight (360,000) (Takedomi et al., 1995). In Experiment II, we wanted to test the efficiency of the pFSH–PVP treatment in a single i.m. injection, changing the administration time of the hormonal preparation in PVP with MW = 40,000, from 48 (Experiment I) to 24 h before the end of progestagen treatment. In effect, this time resulted effective in the induction of superovulation and provided an embryo production slightly higher compared to the control. These results were confirmed
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in Experiment III, where we used PVP having MW = 10,000. In this trial, the importance of treatment time but also the method of administration of pFSH–PVP bolus was emphasized. Injection of pFSH dissolved in PVP 48 h before pessary withdrawal induced a significant delay of estrus onset, a low ovulation rate and a poor embryo production, following either i.m. or s.c. administration. The administration method of pFSH–PVP plays a role in the efficiency of superovulatory treatment. It was reported (Kelly et al., 1997) that pFSH was released into the bloodstream more slowly from a s.c. deposit than from an i.m. site, maintaining higher circulating levels of gonadotrophin over a longer time. Moreover, FSH absorbtion rate is affected by the amount of fat present at the site of injection, as referred by Bo et al. (1994) in a study on cows. The low superovulatory response obtained in ewes treated with pFSH–PVP bolus 48 h before sponge removal could be ascribable to the high circulating levels of the gonadotrophin, due to the administration of 250 IU pFSH all at once, in advance compared to the physiological events regulating ovulation rate. It was demonstrated that in sheep recruitment of follicles occurs after the luteolysis (Driancourt et al., 1985). In our study, administration of the gonadotrophin coincided with the induction of luteolysis by PGF2␣ injection (at 48th hour before sponge removal). Thus, we can deduce that at this time PVP was unable to maintain sufficient pFSH activity to sustain growth of multiple follicles, even if following s.c. administration.The best superovulatory response was obtained following pFSH–PVP treatment 24 h before sponge removal, but performing the single hormone administration by i.m. injection, confirming the findings reported by Dattena et al. (1994) in Sarda ewes. Ewe genotype may also play an important role in ovarian response to the hormone treatment as unsatisfactory results were reported in Romanov ewes (Lajous et al., 1997) as opposed to the positive results in Sarda ewes (Dattena et al., 1994). In conclusion, the results of this study demonstrate the efficiency for superovulation of PVP having molecular weight of 40,000 or 10,000 at 30% w/v as vehicle for treatment with 250 IU pFSH in a single administration, when performed at the 24th hour before the end of progestagen treatment by i.m. injection.
Acknowledgements This research was supported by the Ministero delle Politiche Agricole e Forestali, Special Project RAIZ, Publication No. RZ 257. The authors wish to thank L. Bongermino for statistical processing analysis and L. Basso for technical assistance. References Akbar, A.M., Nett, T.M., Niswender, G.D., 1974. Metabolic clearance and secretion rates of gonadotropins at different stages of the estrous cycle in ewes. Endocrinology 94, 1318–1324. Armstrong, D.T., Evans, G., 1983. Factors influencing success of embryo tansfer in sheep and goats. Theriogenology 39, 7–24. Bo, G.A., Hockley, D.K., Nasser, L.F., Mapletoft, R.J., 1994. Superovulatory response to a single subcutaneous injection of Folltropin in beef cattle. Theriogenology 42, 963–975. D’Alessandro, A., Martemucci, G., Colonna, M.A., Cafueri, C., Toteda, F., 1997. Some effects of adding p-LH in defined amounts to purified pFSH to modify FSH/LH ratios during the superovulatory treatment of anoestrus ewes. Anim. Reprod. Sci. 47, 91–98.
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