Andrea Branca

Recipient synchronization affects viability of vitrified ovine blastocysts

Abstract We assessed the viability of vitrified ovine blastocysts in vitro and in recipient ewes at different degrees of synchronization. Expanded ovine blastocysts obtained from superovulated Sarda ewes were exposed to a vitrification solution at 20 °C according to the following procedures: the blastocysts were added into 200 μl glycerol (1.4 mol) for 5 min, then into 200 μl glycerol (1.4 mol) and ethylene glyco (l 3.6 mol) for 5 min. These embryos were transferred into 25 μl glycerol (3.4 mol) and ethylene glycol (4.6 mol) and loaded into the center of 0.25-ml straws separated from air bubbles by 2 columns of 90 μl sucrose solution (0.5 mol). The straws were plunged immediately into liquid nitrogen (LN 2 ). The basic vitrification and thawing solution was phosphate buffered saline (PBS) supplemented with 20% fetal calf serum (FCS). The embryos were thawed by agitating the straws in a water bath at 20 °C, and expelling them into Petri dishes and mixing them, and placing the embryos into 200 μl sucrose solution (0.25) mol for 5 min. The percentage of hatched blasticysts, after exposed and vitrified-thawed embryos, was 96.8% (3031) and 82.8% (5364), respectively, after 4 d of coculture in TCM 199 medium with 10% FCS in 5% CO 2 humidified atmosphere in air at 39 °C. Vitrified-thawed blastocysts, which re-formed the blastocoelic cavity after 24 h of co-culture as in the in vitro experiment, were surgically transferred into the uterine horn of recipients (2 embryos per ewe) at 6, 7 and 8 d after estrus. The pregnancy and lambing rates in recipient ewes at 6, 7 and 8 d after estrus were 72.7%, 90%, 54.5% and 72.7%, 80%, 45.4%, respectively. Both pregnancy and lambing rates differed significantly (P

Superovulatory response and quality of embryos recovered from anestrus ewes after a single injection of porcine FSH dissolved in polyvinylpyrrolidone.

The effects of a single injection of porcine FSH (pFSH) administered in long acting vehicle on the superovulatory response of milk (Sarda breed) sheep were determined during the anestrous season. The sheep (n=42), synchronized with intravaginal sponges (40 mg fluorogestone acetate -FGA- for 14 d) were submitted 24 h before sponge removal to three different superovulatory treatments. Group 1 (n=16) was treated with a single intramuscular (im) injection of 16 mg of pFHS dissolved in 30 % polyvinylpyrrolidone (PVP); Group 2 (n=12) was injected im with 6, 5, 3 and 2 mg of pFSH every 12 h over 2 d; Group 3 (n=14) was given 800 IU of PMSG and 12 mg of pFSH. All sheep were mated with a fertile ram. Embryos were recovered surgically at Day 7 of sponge removal and graded for the quality according to their morphology. The percentage of good quality embryos recovered was 84% in Group 1, 68% in Group 2 and 77% in Group 3. Data for the onset of estrus, number of corpora lutea (CL), number of unovulated follicles, embryo recovery rate, embryo quality and fertilization rate were recorded for the 3 groups. The onset of estrus, number of CL, number of unovulated follicles, fertilization rate and number of good quality embryos did not differ significantly among the 3 groups. The embryo recovery rate was significantly lower in the group treated with PMSG-FSH (Group 3) than in the 2 other groups. It is concluded that during the anestrous season a single injection im of pFSH results on average in a superovulatory response as good as the more traditional treatments like multiple injections of pFSH and PMSG-pFSH combined.

Comparison of laparoscopic and transcervical insemination with frozen semen in Sarda dairy ewes

Laparoscopic insemination with frozen-thawed semen is currently used for planned matings in the Sarda breeding programme. In order to find a fast and less intrusive artificial insemination (AI) method that could replace laparoscopic insemination, a field comparison of laparoscopic and transcervical techniques was carried out on 200 mature Sarda ewes. After AI, ewes were assigned to teaser and fertile rams for 2 months. Return rates and cumulative (AI + natural mating) lambing rates were recorded over three subsequent 23-day periods. Lambing rates to AI were significantly different (P 0·05) and reached 82% and 74% respectively. Ewes with body condition scores at AI higher than 2·75 showed better overall reproductive performance, but not higher pregnancy rate to AI. Plasma cortisol concentrations, sampled twice, before and after AI, were higher (P < 0·01) in the last sample, suggesting a stress response to insemination. Cortisol levels after AI were lower (P < 0·01) for ewes submitted to transcervical rather than laparoscopic insemination (P < 0·01). However, cortisol levels after AI were no greater than those recorded when ewes were restrained in a milking yoke different from that usually employed. Laparoscopic AI was confirmed as the most suitable technique for insemination offrozen semen in the Sarda breeding scheme.