Laboratorios Calier S.A. PLUSET : MAIN FEATURES   Ramon Calonge Laboratorios Calier S.A. Barcelona - Spain Presentation prepared by Calier and given at AETS Conference August 2004.

2 vials of freeze-dried powder: 500 IU FSH + 500 IU LH each 1 vial of diluent: Normal saline to 20 ml

Background Developed by Serono (Italy) First approval: 1991, in Italy. Later on in USA, Mexico, Argentina, Brazil, New Zealand. 1998: acquired by Laboratorios Calier, S.A. Production plant moved to Barcelona (Spain). Settlement of the industrial extraction and production process. Approval in Colombia, Venezuela, Ecuador. 2003: Approved in 5 EU member states (UK, DE, NL, ES, PT) 2004: Approved in Australia

FSH LH Extraction Method Frozen Pituitary Glands Acetonic phase (2 days) Acetonic powder Alcoholic phase (3 days) Alcoholic powder Extraction phase (4 days) Crude FSH / LH Separation phase (4 days) FSH LH LH FSH

FSH IU FSH LH IU LH Final product Vial filling Lyophilisation Biological analysis (to confirm activity) Biological analysis LH FSH IU LH IU FSH

FSH: Steelman & Pohley method (ovarian weight) Biological analysis FSH: Steelman & Pohley method (ovarian weight) LH: Parlow method (Ascorbic Acid depletion) Described by the WHO The only ones accepted by pharmacopoeial monographs to determine FSH and LH potency of therapeutic preparations Measures biologically active hormones No equivalence with other methods (RIA)

The same proportion of the hMG (human Menopausal Gonadotrophin) LH content The same proportion of the hMG (human Menopausal Gonadotrophin) LH content is known and constant Proportion in weight is  25:1 FSH t ½: 2:30 h LH t ½: 40 min

Recommended Administration Schedule in Holstein-Friesian Dairy Cows - 800 I.U. Day 1 AM PM 3 ml i.m. Start treatment between 9th and 12th day of the cycle, preferably on 11th day Day 2 AM PM 2.5 ml i.m. 2 AI: Inseminate at 12h and 24h of onset of oestrus 3 AI: Inseminate at 12h, 24h and 36h of onset of oestrus Optional: add a dose of GnRH or hCG at 1st AI to induce a contemporaneous ovulation Flushing at 6-8 days (7 days) of the 1st AI Day 3 AM PM 1.5 ml i.m. PGF2 PGF2 Day 4 AM PM 1 ml i.m. Onset of oestrus * 1 ml Pluset: 50 I.U. FSH + 50 I.U. LH

Recommended Administration Schedule in Spain in Beef cows - 700 I.U. Day 1 AM PM 2.5 ml i.m. Day 2 AM PM 2 ml i.m. Beef cows: 600-700 I.U. Beef heifers: 500 I.U. Dairy heifers: 500 I.U. Bos Indicus (zebu): 200-300 I.U. Buffalo: 250 I.U. Sheep: 250-350 I.U. Day 3 AM PM 1.5 ml i.m. PGF2 PGF2 Day 4 AM PM 1 ml i.m. AI Onset of oestrus * 1 ml Pluset: 50 I.U. FSH + 50 I.U. LH

Dairy cows

Use of different commercial FSH preparations in the superovulation of high producing Holstein cows Fuentes, S. and De la Fuente, J. Proceedings, XXI World Buiatrics Congress. Punta del Este, Uruguay, 2000 Total dose: 850 IU Day 1: PRID insertion Day 9: 175 IU + 175 IU Day 10: 125 IU + 125 IU Day 11: 75 IU + 75 IU PRID removal PG (AM and PM) Day 12: 50 IU + 50 IU Holstein Cows, 5-6 years old 3-4 calvings Interval calving-SOV treatment: 5.8-6.5 months Mean milk prod: 27-30 kg/day

Fuentes, S. and De la Fuente, J. Study of different doses of Pluset for the lactating cow superovulation Fuentes, S. and De la Fuente, J. Unpublished data Holstein Cows, 5 years old 2.5 calvings Interval calving-SOV : 6 months Day 1: PRID insertion Day 11: PRID removal PG (AM and PM)

Fuentes, S. and De la Fuente, J. Study of the effect of a similar dose of PLUSET over dairy cows with different parity number Fuentes, S. and De la Fuente, J. Unpublished data Total dose: 850 IU Day 1: PRID insertion Day 9: 175 IU + 175 IU Day 10: 125 IU + 125 IU Day 11: 75 IU + 75 IU PRID removal PG (AM and PM) Day 12: 50 IU + 50 IU Holstein Cows Interval calving-SOV : 4.5-6 months Group A, First calving. n= 12. Group B, Second calving. n= 14. Group C, Third calving. n= 17. Group D, Fourth calving. n= 14.

Superovulation with different doses of PLUSET in relation to parturition number in high production dairy cows Fuentes, S. and De la Fuente, J. IX Jornadas sobre Producción Animal, ITEA, Zaragoza, abril’01 Day 1: PRID insertion Day 11: PRID removal PG (AM and PM) Group A, 800 IU, one calving. n= 15 Group B, 850 IU, two calving. n= 15 Group C, 1000 IU, three calving. n= 14 Interval calving-SOV : 6 months (3-7 m)

Superovulation of Holstein-Friesian cows with reduced dose of FSH-p Bényei B, Barros CWC Arq. Fac. Vet. UFRGS, Porto Alegre, V. 27, n. 1, 1999 (supl.): p. 203 Control: 1000 IU FSH/4 days (n=99) Experiment: 600 IU FSH/4 days (n=83) PG at the 3rd day; AI at 45 and 60h

Arq Bras Med vet Zootec Vol 52, n 4, Belo Horizonte Aug 2000 Effect of superovulation on performance of bovine embryo donors imported from temperate zone to tropical climate during the first two years of adaptation Bényei B, Barros CCW Arq Bras Med vet Zootec Vol 52, n 4, Belo Horizonte Aug 2000 Holstein-Friesian heifers imported pregnant from Hungary to Brazil In the 2nd year were superovulated with Folltropin or Pluset (600 IU / 4 days) FSH in decreasing doses for 4 days, PG at 3rd day, AI at 60-72h

Heifers

Superovulation in cattle: effect of FSH type and methods of administration on follicular growth, ovulatory response and endocrine patterns Kelly P., Duffy P., Roche J.F. and Boland M.P. Animal Reproduction Science 46 (1997): 1-14 Group SF: (n=23) Single Folltropin. 270 mg NIH-FSH-P1 in a single s.c injection. Group MF: (n=23) Multiple Folltropin, 270 mg in 4 days Group SP: (n=22) Single Pluset, 1.000 IU in a single s.c injection Group MP: (n=22) Multiple Pluset, 1.000 IU in 5 days

Effect of timing of oestradiol benzoate injection relative to gonadotropin treatment on superovulatory response, and on embryo yield and quality in beef heifers Yaakub H, Duffy P, O’Callaghan D, Boland MP Animal Reproduction Science (1998) 52: 191-204 Group I: only CIDR for 9 days Group II: 5 mg ODB at CIDR insertion Group III: 5 mg ODB 1.5 days after CIDR insertion Group IV: 5 mg ODB 3 days after CIDR insertion Sov started 6.5d after CIDR insertion 3 doses: 400, 600, 800 IU Pluset in 8 equal i.m injections each 12h RESULTS: No interaction between treatment and dose of Pluset Combination of ODB and CIDR do not improve embryo yield best results achieved with CIDR only

Mantovani R et al - 12nd Meeting AETE - Lyon, 1996: 168 Dose effect of porcine gonadotropin extract containing FSH and LH on superovulatory response and fertility of Friesian heifers Mantovani R et al - 12nd Meeting AETE - Lyon, 1996: 168 60 cycling heifers, 13-16 months Dose in 9 i.m doses, each 12h (4 1/2 days), giving 35%, 25%, 20%, 15% and 5% of the total dose Results: Optimal response with 500 IU FSH, with no effects on subsequent reprod. Performance higher dose affects embryo yield and quality, but not fertility

Beef cows

Effects of prepartum lipid supplementation on FSH superstimulation and embryo quality in multiparous beef cows Bader JF et al Theriogenology, v 57, n 1, 2002: 760 40 mature cows, prepartum lipid supplement 40 days prior to calving WSB: whole soybean supplement GnRH-7 days- GnRH- 7 days- PG: 28 cows responded Superovulation started days 8-10 cycle (60d postpartum) 600 IU at decreasing doses for 4 days, PG: 96h and 108h after start treatment 27 cows showed oestrus: AI 0, 12 and 24h after onset oestrus

Evaluation of an estrus synchronization protocol prior to superovulation in beef cows Kojima FN et al Theriogenology, v 57, n 1, 2002: 766 24 crossbred non-lactating beef cows Protocol 7-11 synch (n=11) or control (n=13) 7-11 Synch: MGA for 7 days + PG 7th day + GnRH 11th day Control synchronised with 2 PG 14 days apart 2 days after GnRH: 600 IU FSH, decreasing doses twice daily, for 4 days PG: 2 adm. on the last day FSH - 5th Day: PG, AI at 48h and 60h

Zebu (Bos indicus)

Total Doses Recommended in Brazil Breed Age Minimum Maximum Nelore Heifer 220 UI 260 UI Cow 250 UI 350 UI Simmental 280 UI 320 UI 400 UI Holstein 420 UI 500 UI Crossbred Zebu/European 230 UI

Efficiency of two different hormonal products for superovulation of Nelore cows Diniz EG et al Rev. Bras. Reprod. Anim., v.23, n.3, 1999: 319-321 Comparison of 250 IU Pluset vs. 20 mg NIH Folltropin in Nelore Cows (Bos indicus) FSH in decreasing doses, in 4 days. PG: 2 doses (AM-PM) of the 3rd day AI: 12 + 24h post-oestrus

Rev. Bras. Reprod. Anim., v.26, n.3, 2002: 246 - 248 Infuence of age on the superovulatory response of Nelore donors: preliminary results Oliveira MAL et al Rev. Bras. Reprod. Anim., v.26, n.3, 2002: 246 - 248 Comparison of superovulatory response of 100 Nelore donors allocated by age: GI: 2-5 years; GII: 6-8 years; GIII: 9-12 years; GIV: >12 years 400 IU FSH in decreasing doses, in 4 days. PG: on the 3rd day. AI: 12 + 24h post-oestrus Group N cows Total structures Viable embryos Degenerate Ova 2-5 y 25 15.8 ± 10.9 9.8 ± 8.4 2.3 ± 2.2 2.9 ± 3.7 6-8 y 18.5 ± 12.3 11.5 ± 10.0 3.3 ± 3.7 4.0 ± 4.8 9-12 y 13.6 ± 13.6 6.5 ± 5.1 3.1 ± 2.9 4.3 ± 5.5 > 12y 9.2 ± 5.0 4.0 ± 2.6 1.6 ± 2.0 3.6 ± 4.3

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