Abstract
This study was designed to optimize the multiple ovulation and embryo transfer (MOЕТ)
technique and semen cryopreservation in indigenous sheep for accelerating genetic
improvement. In MOET study, all ewes were synchronized with 45 mg fluorogestone acetate
(FGA) sponge for 12 days and donor ewes were superovulated by injecting ovine or porcine
follicle stimulating hormone (oFSH or pFSH). Two flushing media; home-made [phosphate
buffered saline (PBS) plus 1% sheep serum] and EmcareTM, and two holding media; homemade (PBS plus 20% sheep serum) and EmcareTM were used for flushing and holding
embryos. The embryo recovery and transfer were performed by modified semi-laparoscopic
method on Day 5/6. Number of corpora lutea (CL) was counted. Progesterone (P4)
concentration of recipient ewes was assayed at day of embryo transfer (ET) and maternal
recognition of pregnancy (MRP) and grouped as ≤1, >1 - 6, >6 - 12 and >12 ng/ml,
respectively. For semen cryopreservation and Artificial Insemination (AI) study, 16 rams
were evaluated for semen quality. Three diluents (D1: tris-egg yolk, 7% glycerol; D2: tris-egg
yolk, 5% glycerol and D3: Triladyl®) and 4 freezing techniques (F1: +5°C to -140°C; F2: +5°С
to -80°C and -80°C to -140°C, F3: +5°C to -80°C, -80°C to -120°C and -120°C to -140°C and, F4:
+5°C to -100°C and -100°C to -140°C) were used for freezing ram semen. Three thawing rate 37°C/12 sec, 37°C/20 sec and 37°C/60 sec were compared. LAP-AI was performed using
two different sperm doses (100×106 and 50×106) and diluents (D3 and D2). TC-AI was
performed using two different sperm doses (200×106 and 100×106), diluents (D3 and D2) and
inseminated single or double. The oestrus response rate was 100% with 45 mg FGA, and had
significantly (P<0.001) earlier onset of oestrus with pFSH than oFSH (23.41±0.50 h vs
26.15±0.46 h). The CL/donor was significantly (P<0.04) higher with pFSH than oFSH (14.18±1.63 vs 10.25±0.89). More embryos were recovered/donor with pFSH than oFSH
(11.82±1.48 vs 8.81±0.86). The embryo recovery rate was higher with oFSH than pFSH
(85.04±2.93% vs 82.75±3.31%). The proportion of morula stage and grade 1 embryos was
higher with both oFSH and pFSH (86.52% and 97.16% vs 84.58% and 90.55%). The proportion
of grade 1 embryos was significantly (P<0.008) higher with PBS plus 1% sheep serum than
EmcareTM (96.3% vs 90.5%). The pregnancy rate (71.4 - 81.0% vs 65.2-73.9%) was higher with
EmcareTM than PBS plus 20% sheep serum, whereas lambing rate (76.5% vs 64.7%), embryo
survival rate at lambing (52.2% vs 49.9%) and lamb survival rate at weaning (75.0% vs 72.2%)
were higher after PBS plus 20% sheep serum than EmcareTM, The pregnancy rate (71.9-75%
vs 59.4 - 68.8%), lambing rate (68.8% vs 56.2%) and embryo survival rate (42.5% vs 66.7%)
were significantly higher (P<0.001) with P4 group>1-6ng/ml (3.44±0.18 ng/ml vs 4.57±0.19
ng/ml) at day of ET and MRP, respectively. There was increased probability of lambing
(P<0.04) and number of lambs (P<0.009) with increased concentrations of P4 at MRP. The
selected 62.5% (10/16) best rams had the international standard semen parameters. The postthaw sperm parameters differed significantly (P<0.05) between the diluents and freezing
techniques. The diluents (D3 and D2) and freezing technique (F3 and F4) resulted in
consistent post-thaw sperm quality. The sperm motility, viability, plasma membrane
integrity and acrosome integrity were above 50%, 65%, 60% and 65%, respectively. The
thawing rate 37°C/20 sec produced the best results. With LAP-AI, the pregnancy rate
(72.2%), lambing rate (100%) and multiple birth rate (66.7%) were higher with 100×106/D3
and 50×106/D2, respectively. With TC-AI, the pregnancy rate was higher (35.3% vs 26.1%)
with 200×106/double AI/D3 and 200×106/double AI/D2. The lambing rate was 100%. The
multiple birth rate were higher (100% and 75%) with 200×106/double AI/D2 and
200×106/single AI D3 or D2, respectively. Objectively, the selected gonadotrophins and the
refined modified semi-laparoscopic method resulted in a consistent response and increased
embryo recovery rate, indicating optimization of MOET. Ram semen cryopreservation was
developed, and LAP-AI and TC-AI were optimized.