Effect of estradiol supplementation on superovulation in Swamp buffalo

The effect of estradiol-17beta (E(2)) supplementation on superovulation with (PMSG) or (FSH) was investigated in Swamp buffalo. Sixty-eight buffalo were treated in seven groups. Group 1 served as control and was superovulated by standard PMSG or FSH treatment used in routine bovine embryo transfer protocols. Group 2 was superovulated by standard PMSG regimen plus two injections of E(2) at a 48 h interval beginning one day before the onset of gonadotropin treatment (short-term supplementation) for a total dosage of 2.5 mg E(2); Groups 3 and 4 received the same regimen as Group 2, but in doses of 5.0 and 7.5 mg E(2), respectively. Group 5 received the standard FSH regimen (40% LH). Group 6 received short-term E(2) (7.5 mg) supplementation of FSH-p. Group 7 was superovulated by standard FSH regimen (40% LH) plus three injections of E(2) at 48-72 h intervals beginning five days before the onset of gonadotropin treatment (long-term supplementation) for a total dosage of 7.5 mg E(2). The number of corpora lutea (CL) and follicles >/= 8 mm in diameter were recorded by palpation per rectum and after slaughter. The mean numbers of CL and follicles were 0.99, 5.8, 8.0, 10.6, 4.0, 3.9, 8.1 and 0.25, 6.8, 6.2, 6.2, 1.6, 0.0, 4.1 for Groups 1, 2, 3, 4, 5, 6, 7, respectively. In Group 7, the rates of nonsurgical and postmortem embryo recovery were 46 and 90.4%, respectively and 54.4% of the collected ova were fertilized. These results indicate the possibility of producing viable embryos in buffalo by using E(2) supplementation for the gonadotropin treatment.     

Use of PMSG antiserum in superovulated cattle?

Two Pregnant Mare Serum Gonadotrophin (PMSG) antisera were tested in 174 dairy cows that were superovulated with PMSG and were then given prostaglandin at 60 hours after PMSG. At 48 hours after injection of prostaglandin, the cows were given either PMSG antiserum (monoclonal (n=56) or polyclonal (n=57)), or saline as control (n=61). Ova (n=1,206) were recovered either nonsurgically or after slaughter. Of these, 757 were evaluated morphologically to be transferable embryos. A proportion of these embryos (n=295 from 52 flushed donors) were transferred to synchronized recipients and the pregnancy results were recorded. The reproductive function of 37 flushed donors was followed for 6 months after superovulation. No significant effect of the PMSG antisera could be demonstrated in any of the parameters studied (i.e., ovulation rate, number of follicles at collection, total yield of ova, fertilization rate, number of transferable embryos, pregnancy results after transfer of embryos, or period required by the donor cows for restitution of reproductive function after superovulation and recovery). It is concluded that use of PMSG antiserum did not improve the embryo yield in terms of the number and quality of transferable embryos or enhance normalization of reproductive function of the donor in the 6-month period after superovulation. Therefore, in an embryo transfer operation, the routine use of PMSG antiserum in a PMSG superovulation regimen in cattle is not recommended.     

Melatonin treatment of embryo donor and recipient ewes during anestrus affects their endocrine status, but not ovulation rate, embryo survival or pregnancy

Thirty-two Border Leicester x Scottish Blackface ewes that lambed in March were individually penned with their lambs from April 16th and given daily an oral dose of 3 mg melatonin at 1500 h (Group M). A further 32 acted as controls (Group C). Within each group half were used as embryo donors (Group D) following superovulation and half received embryos (Group R) following an induced estrus. Prior to weaning on 21 May ewes received ad libitum a complete diet providing 9 megajoules (MJ) of metabolizable energy and 125 g/kg crude protein. Thereafter each received 1.6 kg of the diet daily. In early June each ewe received an intravaginal device (300 mg progesterone) inserted for 12 d. Donors were superovulated with 4 i.m. injections of porcine FSH 12 h apart, commencing 24 h before progesterone withdrawal. Ovulation in recipients was induced with 800 IU PMSG injected i.m. at progesterone removal. Donor ewes were inseminated 52 h after progesterone withdrawal. Embryos were collected 4 d later and transferred to recipients. Melatonin suppressed plasma prolactin (P < 0.001) and advanced estrus (P < 0.05) and timing of the LH peak (P < 0.05). These events also occurred earlier in donors than in recipients (P < 0.01). Mean (+/- SEM) ovulation rates for melatonin-treated and control donors were 5.5 +/- 0.71 and 4.7 +/- 0.66, respectively (NS). Corresponding recipient values were 3.3 +/- 0.40 and 3.4 +/- 0.39 (NS). Mean (+/- SEM) embryo yields were 2.9 +/- 0.64 and 2.6 +/- 0.73 for melatonin-treated (n = 15) and control (n = 16) donors, respectively, and for the 12 ewes per treatment that supplied embryos, corresponding numbers classified as viable were 2.7 +/- 0.47 and 2.3 +/- 0.61 (NS). Following transfer, 57% of embryos developed to lambs when both donor and recipient received melatonin, 86% when only the donor received melatonin, 91% when only the recipient received melatonin, and 67% when neither received melatonin (NS). Thus, embryo survival following transfer was not improved by treating recipients with melatonin. Gestation length and lamb birthweights were unaffected by melatonin. Unlike nonpregnant control ewes, melatonin-treated recipients that failed to remain pregnant sustained estrous cyclicity following embryo transfer.     

Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P < 0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P > 0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.     

Superovulation treatments and embryo transfer in Angora goats

A high incidence of early luteal regression after PMSG superovulation was associated with low recovery of embryos from reproductive tracts of Angora goats flushed later than Day 5 after onset of oestrus. Embryos were successfully recovered (mean 7.9/female) by flushing on Days 2-5. Mean ovulation rate after an FSH regimen (16.1 +/- 0.8) was significantly higher than that after a single injection of PMSG (10.8 +/- 1.2). Fertilization rate and survival of embryos following transfer to naturally synchronized recipient feral goats did not differ between the two gonadotrophin regimens: the mean number of kids born to 47 donors treated with FSH (7.5 +/- 0.6) was significantly greater than that to 28 donors treated with PMSG (4.8 +/- 0.6). Irrespective of hormonal treatment, the numbers of embryos recovered and of kids born were correlated with ovulation rate (r = 0.82, P less than 0.001 for both). Embryo survival was influenced by ovulation rate in recipients, with 52%, 63% and 75% of transferred embryos being carried to term by recipients with 1,2 and 3 CL, respectively (P less than 0.01). More embryos survived (65%) when 2 embryos were transferred to each recipient than when 1 (51%) or 3 (48%) were transferred. In recipients receiving 2 embryos, survival was significantly improved by transfer of both embryos to the same oviduct (70%) than when one was transferred to each oviduct (62%). The percentage survival of embryos was optimal when oestrus of recipients was synchronized within +/- 1 day of oestrus in donors.     

SUPEROVULATION AND SUPERPREGNANCY IN THE GOLDEN HAMSTER*

Pregnant mare serum gonadotropin (PMSG) treatment given in the morning or afternoon on any day of the four-day estrous cycle and human chorionic gonado tropin (HCG) given two days later successfully induced superovulation in the golden hamster. The minimum interval between PMSG and HCG necessary to obtain consistent superovulation was approximately 44 hr. The lowest ovulation rate was obtained following PMSG treatment on the afternoon of day 4 despite the fact that this time coincides with the maximum endogenous FSH level, necessary for the maturation of the next crop of follicles destined to ovulate. Thirty-eight to one-hundred percent of superovulated females in four different treatment groups became superpregnant after natural mating. Some treated females exhibited two consecutive nights of estrus with ovulation apparently occurring during the second night. Superpregnant females delivered “super” size litters, up to 27 live-born pups. The ultimate litter size appeared to be established after day 3 and prior to day 8 of superpregnancy. A one-day extension of the normal 16-day gestation period was observed in 31% of superpregnancies. Unilateral pregnancies were observed at autopsy in 44% of treated females which received the high dose of PMSG (30 IU). The progeny of superovulated females reproduced normally at maturity. The results indicate that ova from superovulated female hamsters are capable of full normal development.     

Follicular development and superovulation response in cows administered multiple FSH injections early in the estrous cycle

To determine whether follicular development, superovulation and embryo production were affected by the absence or presence of a dominant follicle, cows were administered injections of FSH twice daily in the early (Days 2 to 6, estrus = Day 0) or middle stage (beginning on Day 10 or 11) of the estrous cycle. Treatment with FSH early in the cycle stimulated follicular development in 83 to 100% of all cows from 4 groups evaluated at different times after PGF2alpha treatment on Days 6 and 7. However, the proportion of cows with > 2 ovulations varied from 31 to 62.5%, indicating that induction of follicular development may occur in the absence of superovulation. When compared with cows treated in the middle of the cycle, no differences were observed in the proportion of cows with > 2 ovulations (31 vs 20%), ovulation rate. (26.0 +/- 6.3 vs 49.6 +/- 25.8), production of ova/embryos (13.3 +/- 3.2 vs 14.4 +/- 3.4), or the number of transferable embryos (8.0 +/- 3.6 vs 5.4 +/- 1.5; early vs middle, respectively). The proportion of the total number of embryos collected that were suitable for transfer was greater (P<0.01) in cows treated early in the cycle (60%) than at midcycle (37.5%). The diameter of the largest follicle observed by ultra-sound prior to initiation of FSH treatment in the early stage of the cycle (10.0 +/- 2.0 mm) was smaller (P<0.05) than at midcyle (16.8 +/- 1.3 mm). These results demonstrate that superinduction of follicular development is highly consistent after FSH treatment at Days 2 to 6 of the cycle and that superovulation and embryo production are not less variable than when FSH is administered during the middle of the cycle. However, superovulation in the early stage of the cycle may increase the proportion of embryos suitable for transfer.     

Ovarian response, embryo recovery and results of embryo transfer in a Hungarian native pig breed

The objective of the study was to use embryo transfer (ET) for propagation of the Swallow Belly Mangalica population. Mangalica is a native Hungarian pig breed adapted to extreme climate and housing conditions and distinguished for excellent meat and fat quality. However, due to their weak reproductive characteristics and relatively high fat proportion, Mangalica pigs have been replaced by modern breeds. Now, there is an increased interest again to safeguard the properties of this breed. We conducted two experiments. First, we used a total of 18 puberal Mangalica gilts to determine an optimal superovulatory treatment. Following estrus synchronization with Regumate, we injected gilts with either 750, 1000 or 1250 IU PMSG, followed by 750 IU hCG 80 h later. We scanned ovaries endoscopically 3 days after hCG administration. The application of 1000 and 1250 IU PMSG resulted in a higher rate of ovulation compared to 750 IU (24.2 +/- 3.6 and 21.0 +/- 2.3 vs. 13.7 +/- 2.7 P<0.05). The number of follicular cysts increased after administration of 1250 IU PMSG compared to 750 and 1000 IU (2.0 +/- 1.3 vs. 0.3 +/- 0.7 and 0.2 +/- 0.3, P<0.05). Thus, we chose 1000 IU PMSG for further stimulation of Mangalica gilts. In the second experiment, we induced superovulation in 10 Mangalica donor gilts by 1000 IU PMSG and 750 IU hCG. Gilts were fixed-time inseminated, and then five days later embryo collection was carried out surgically (n=6) or endoscopically (n=4). Out of the 187 ova recovered, 92.5% were at the morula/blastocyst stage. The embryo recovery rate was higher following surgical flushing than following endoscopy (91.5 +/- 4.4% vs. 71.4 +/- 12.7%, P<0.05). Altogether 143 embryos were transferred surgically or endoscopically into 8 Landrace recipients. Surgical and endoscopic transfer of Mangalica embryos into Landrace gilts resulted in pregnancies in 3 and 2 gilts, respectively; thus the overall farrowing rate was 62.5%. The birth of 59 Mangalica piglets from 5 embryo recipients equals an average litter size of 11.8 +/- 1.3, which is two times larger than usual in this breed. Therefore, we concluded that an appropriate inter-breed ET program is a suitable tool to propagate the endangered Mangalica breed.     

Ovulation rate, zygote recovery and follicular populations in FSH-superovulated goats treated with PGF(2alpha) and/or GnRH

Follicular development and ovulation were examined in superovulated Nubian and Nubian-cross dairy goats following prostaglandin F(2alpha) (PGF(2alpha)) and/or gonadotropin releasing hormone (GnRH) treatment. Estrus was synchronized with Synchromate-B((R)) implants. Superovulation was induced with follicle stimulating hormone (FSH) and augmented with GnRH and/or PGF(2alpha). The PGF(2alpha) treatment was administered on Day 2 of superovulation. Implants were removed from all goats on Day 3 of superovulation. The GnRH treatment was administered 24 h after implant removal. All does were exposed to fertile males for 48 h at the time of GnRH injection. Surgical embryo recovery and ovarian response evaluation were conducted 64 to 78.5 h after implant removal. The number of ovulations was higher with GnRH treatment (18.5 +/- 7; x +/- SEM) than that in the controls (5.3 +/- 4.1; P < 0.05). There were fewer follicles in the GnRH-treated does than in the untreated does (10.9 +/- 2.9 vs 22.1 +/- 3.2; P < 0.05). The number of follicles smaller than 4 mm in diameter (5.8 +/- 0.8) did not differ between treatments. The GnRH-treated does had fewer 4- to 8-mm follicles (4.2 +/- 2.0 vs 9.1 +/- 1.6; P < 0.05) and fewer follicles larger than 8 mm (0.7 +/- 1.4 vs 7.3 +/- 1.6; P < 0.01) than the controls. Predicted times for 1- and 2-cell embryo recoveries were 68.5 and 73.7 h following implant removal, respectively. This study demonstrates that GnRH is an effective supplement used with FSH superovulation regimens in dairy goats. Moreover, GnRH provides for enhanced early embryo collection for DNA microinjection studies.     

Successful in vitro culture of early cleavage stage embryos recovered from superovulated red deer (Cervus elaphus)

Three separate embryo culture systems were evaluated for their ability to support development of early cleavage stage red deer (Cervus elaphus ) embryos: ligated sheep oviducts (Treatment A); cervine oviduct epithelial monolayer in TCM 199 + 10% deer serum (Treatment B); synthetic oviduct fluid + 20% human serum at 7% O(2) atmosphere (Treatment Q. In addition, 2 superovulation protocols were compared for their efficacy in producing early cleavage stage embryos. Twenty red deer (2 to 7 yr old) were synchronized in April with intravaginal CIDR devices for 12 d. All animals received a total of 0.4 units of ovine FSH administered in 8 equal doses, 12 h apart, beginning 72 h before removal of CIDR devices. The deer additionally received 200 IU PMSG, either with the first FSH injection (Group 1, n = 10) or with the last FSH injection (Group 2, n = 10). Hinds were placed with fertile stags following withdrawal of CIDR devices. Ova were collected by surgical recovery 63 h post CIDR removal. At the time of collection, animals in Group 2 had a significantly greater mean (+/- SEM) ovulation rate (11.2 +/- 2.4 vs 5.3 +/- 2.4), with more animals responding to treatment (>1 ovulation), than the animals in Group 1 (10/10 vs 4/10). Late in the breeding season (June), 10 additional red deer (Group 3, Experiment 2) were superovulated using the same protocol as for the deer in Group 2, with ova collection advanced by 24 h. Mean (+/- SEM) ovulation rate was 6.4 +/- 1.2 with 9 10 animals responding. Ova recovery did not differ among the groups (range 73 to 87%). Superovulation treatment did not affect cultured embryo development to the morula/blastocyst stage. Furthermore, there was no difference among the 3 culture systems in their support of development either to the morula (range 50 to 58%) or to the blastocyst (range 22 to 26%) stage. After laparoscopic transfer of 4 morula/blastocyst embryos to recipient red deer (2 from Treatment B and 2 from Treatment C) 2 live calves were born from embryos cultured in Treatment B.     

The effects of superovulation of donor sows on ovarian response and embryo development after nonsurgical deep-uterine embryo transfer

This study aimed to evaluate the effectiveness of superovulation protocols in improving the efficiency of embryo donors for porcine nonsurgical deep-uterine (NsDU) embryo transfer (ET) programs. After weaning (24 hours), purebred Duroc sows (2–6 parity) were treated with 1000 IU (n = 27) or 1500 IU (n = 27) of eCG. Only sows with clear signs of estrus 4 to 72 hours after eCG administration were treated with 750 IU hCG at the onset of estrus. Nonhormonally treated postweaning estrus sows (n = 36) were used as a control. Sows were inseminated and subjected to laparotomy on Days 5 to 6 (Day 0 = onset of estrus). Three sows (11.1%) treated with the highest dosage of eCG presented with polycystic ovaries without signs of ovulation. The remaining sows from nonsuperovulated and superovulated groups were all pregnant, with no differences in fertilization rates among groups. The number of CLs and viable embryos was higher (P < 0.05) in the superovulated groups compared with the controls and increased (P < 0.05) with increasing doses of eCG. There were no differences among groups in the number of oocytes and/or degenerated embryos. The number of transferable embryos (morulae and unhatched blastocysts) obtained in pregnant sows was higher (P < 0.05) in the superovulated groups than in the control group. In all groups, there was a significant correlation between the number of CLs and the number of viable and transferable embryos, but the number of CLs and the number of oocytes and/or degenerated embryos were not correlated. A total of 46 NsDU ETs were performed in nonhormonally treated recipient sows, with embryos (30 embryos per transfer) recovered from the 1000-IU eCG, 1500-IU eCG, and control groups. In total, pregnancy and farrowing rates were 75.1% and 73.2%, respectively, with a litter size of 9.4 ± 0.6 piglets born, of which 8.8 ± 0.5 were born alive. There were no differences for any of the reproductive parameters evaluated among groups. In conclusion, our results demonstrated the efficiency of eCG superovulation treatments in decreasing the donor-to-recipient ratio. Compared with nonsuperovulated sows, the number of transferable embryos was increased in superovulated sows without affecting their quality and in vivo capacity to develop to term after transfer. The results from this study also demonstrate the effectiveness of the NsDU ET procedure used, making possible the commercial use of ET technology by the pig industry.     

Factors affecting the yield of viable embryos by superovulated Holstein-Friesian cows

GnRH treatment (250 ug) 48 h after prostaglandin F(2alpha) in 40 superovulated cows induced a release of LH (increment > 5 ng/ml) in only 13 of the older cows. Eleven of these cows did not yield viable embryos. Thirty-two of 75 cows had preovulatory surge levels of LH 48 h after prostaglandin treatment. Plasma progesterone concentrations were determined in 140 cows at the time that superovulation was initiated. Eighty-four of these donors were superovulated with 40 mg of FSH and 56 donors with 48 mg of FSH. There was no relationship (P > 0.05) between the concentration of progesterone at the start of superovulation with either ovulation rate determined by palpation per rectum or the number of viable embryos per flush. These parameters were also unaffected (P > 0.05) by age of the donor or the dose of FSH. In another group of donors, treatment with 40 mg FSH was compared over a 3-d (n = 28) and a 4-d (n = 18) interval. The donors treated with FSH over a 3-d period had similar ovulation rates but yielded less viable embryos (1.5 v 5.8, P < 0.05). The fertility rate of 33 cows, inseminated 60 and 72 h after prostaglandin, was comparable to the fertility rate of 18 cows inseminated at 60, 72 and 84 h after prostaglandin treatment.     

Superovulating cows with follicle stimulating hormone and pregnant mare's serum gonadotrophin

A total of 511 embryos was recovered non-surgically from nearly 100 superovulated or untreated donors. Superovulation with FSH-LH resulted in more corpora lutea, recovered ova, and pregnancies (P<.01) than superovulation with PMSG. No differences were observed in numbers of ovulations, embryos recovered, or pregnancies per donor when prostaglandin F_2α was given to donors 2 versus 3 days following gonadotrophin treatment.Pregnancy rates of 12, 31, 58, and 63% were obtained from groups of embryos classified morphologically as poor, fair, good, and excellent (P<.05). Morphologically normal embryos collected and transferred at 5 to 6 days of gestation resulted in more (P<.05) fetuses (75%) than morphologically normal embryos at 8 to 9 days of gestation (56%), but neither was significantly different from morphologically normal embryos at 6.5 to 7.5 days of gestation (61%). There was no difference (P>.05) between pregnancy rates when retarded embryos from untreated donors (12%) were compared to retarded embryos from superovulated donors (22%). However, a higher proportion of morphologically normal embryos from untreated donors developed into fetuses (71%) than did morphologically normal embryos from superovulated donors (59%, P<.05).     

Repeat superovulation, non-surgical embryo recovery, and surgical embryo transfer in transgenic dairy goats

We investigated the capability of repeat superovulation and non-surgical embryo retrieval, coupled with surgical embryo transfer, to expedite the production of transgenic progeny from transgenic founder dairy goat does. In addition, we compared embryo yields, number of embryos transferred per recipient, pregnancy rates, and offspring born during both the traditional (September-December) and non-traditional (January-May) breeding seasons. Although there were no significant differences, there were numerically more transferable embryos recovered per flush (3.5+/-0.9 vs. 2.4+/-0.9 embryos; mean+/-S.E.M.) and increases in both the proportion of recipients that were pregnant (83 vs. 69% pregnant) and offspring born from total embryos transferred (67 vs. 53% offspring) during the traditional versus the non-traditional breeding season. The transfer of one, two or three embryos did not significantly affect the proportion of pregnant recipients during either season. However, there was a difference (P<0.05) in the proportion of offspring produced for one versus two embryo transfers (89 vs. 44% offspring, respectively) during the non-traditional breeding season. Overall, 14 transgenic offspring were produced from 54 total offspring born, and the kidding interval was reduced to <3 months for six of the seven transgenic does. In summary, repeat superovulation and non-surgical embryo retrieval, coupled with surgical embryo transfer, expedited the production of progeny from transgenic founder does.     

Superovulation of rabbits with FSH alters in vivo development of vitrified morulae

Morulae were flushed from the oviducts and uteri of New Zealand White (NZW) rabbits superovulated with either 6 (3 d) or 8 (4 d) injections of FSH and from non-superovulated controls. The percentages of embryos recovered from 4 d (100%, n = 8) donors was significantly higher (P < 0.01) than that of 3 d (76%, n = 16) and control (87%, n = 22) donors. Overall, fertilization rates were significantly lower for the 3 d embryos (P < 0.01). Most (86 to 90%) morulae were morphologically suitable for vitrification in an ethylene glycol-based solution. Following storage in liquid nitrogen, morulae were rapidly thawed and transferred to the uteri of pseudopregnant recipients. The total number of kits born for the 3 d, 4 d, and control groups was 40, 61 and 48, respectively. The percentage of live kits from morulae transferred was significantly lower for the 3 d (20%, n = 201) than either the 4 d (36%, n = 169; P < 0.01) or the control (31%, n = 157; P < 0.05) group. The mean number of kits born/recipient for the 3 d (2.4 +/- 2.9), 4 d (4.7 +/- 3.5), and control (3.0 +/- 2.2) protocols did not differ (P > 0.05). The estimated overall efficiency of producing kits based on normal morulae collected for control and 4 d groups, however, was nearly two-fold that for females given 6 FSH treatments. We conclude that the 4 d FSH superovulation regimen enhances the efficiency of rabbit reproductive biotechnology after embryo cryopreservation. These findings have important implications for rabbit colony management using embryo cryopreservation.     

In vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatment in rabbit does

This study aimed to evaluate the in vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatments in rabbit does. Ninety-six nulliparous does were randomly subjected to consecutive superovulation treatments with eCG (20 IU/kg body weight intramuscularly (i.m.), eCG group), FSH (3 x 0.6 mg/doe at 24 h intervals i.m., FSH group), or without superovulation treatment (control group). Does were artificially inseminated 3 days later and ovulation was induced immediately by hCG (75 IU/doe intravenous). Seven experimental groups were differentiated: first FSH and eCG treatment, second FSH and eCG treatment, eCG-interchanged group (does with previous FSH treatment), FSH-interchanged group (does with previous eCG treatments) and control group. Embryos were collected in vivo by laparoscopy 76-80 h post-insemination in the first and second recovery cycles and post mortem in the third recovery cycles. The ovulation rate was significantly higher in does treated with the first-FSH than in those treated with eCG or in control does (25.2+/-2.0 versus 19.2+/-1.4 to 11.0+/-1.5, and 12.2+/-1.2, first-FSH, first-eCG to second-eCG and control groups, respectively, P < 0.05). Significant differences were observed in the total recovery influenced by ovulation rate in each group (20.3+/-2.2 to 9.4+/-1.2, first-FSH to control groups). Embryo donor rate (donor with at least one normal embryo) was similar among groups with an overall of 75.1%. The number of normal embryos recovered per doe with at least one normal embryo increased significantly in relation to ovulation rate (17.7+/-2.2 to 8.41+/-3, first-FSH and control groups). The vitrification of embryos negatively affected their in vitro development to hatched blastocyst in all groups (88.1% versus 48%, P > 0.05). However, after embryo transfer, this negative effect was only observed in superovulated vitrified embryos (16.8 and 12.8% versus 39.4% total born rate from eCG, FSH and control vitrified groups, P < 0.05). Results indicated that the primary treatments with eCG or FSH increased the number of normal embryos recovered per donor doe, but these embryos are more sensitive to vitrification protocols.     

Fertilization and embryo recovery rates in superovulated chios ewes after laparoscopic intrauterine insemination.

Forty superovulated dairy ewes of the Greek Chios breed were used in an experiment to evaluate the efficiency of laparoscopic intrauterine insemination on fertilization and embryo recovery rates as well as embryo quality. Estrus was synchronized by intravaginal progestagen impregnated sponges and superovulation was induced by administration of 8.8 mg o-FSH i.m. following a standard 8 dose protocol. A small volume (0.3 mL) of diluted fresh ram semen was deposited in each uterine horn 24 to 28 h after onset of the estrus by a laparoscopic technique. The animals were allocated randomly into two groups (Group A and B) of 20 animals each. In Group A, embryos were recovered 18 to 24 h after the intrauterine insemination and in Group B on Day 6. The average number of corpora lutea was 12.8 +/- 1.2 and 11.5 +/- 1.1 (+/- SEM); the overall embryo recovery was 66.4% and 57% and the percentage of recovered fertilized ova was 81% and 82.8% in Groups A and B, respectively. More fertilized ova were collected per ewe from Group A (P < or = 0.1). Results indicated that in Chios breed, superovulation using homologous FSH combined with laparoscopic AI leads to good ovarian response with satisfactory results in fertilization, embryo recovery and quality of embryos. This could lead to improved and more efficient methods for obtaining large numbers of high quality oocytes and embryos for embryo transfer programs which could contribute to genetic improvement and increase of the population size.     

The effect of two different levels of progesterone priming on the response of ewes to superovulation

The use of either 1 or 3 controlled internal drug release (CIDR) devices for progesterone priming in ewes (n=11) superovulated with 1500 IU pregnant mare serum gonadotrophin (PMSG) at 28 hours prior to CIDR device withdrawal was investigated in relation to the stages of development and viability of the ova produced. Progesterone levels in the ewes (n=6) treated with 3 CIDR devices were significantly higher (P<0.01) during the 11 days of insertion than in those (n=5) treated with 1 CIDR device (7.3 vs 3.3 ng/ml) over the same period. However, following superovulation, the mean (+/-SEM) ovulation rates were similar for both groups (8.2 +/- 1.7 vs 10.2 +/- 1.5). The number of ova (M+/-SEM) recovered by laparoscopy 5 days after insemination was 4.2 +/- 1.0 for ewes treated with 3 CIDR devices and 7.0 +/- 1.1 for those treated with 1 CIDR device (P<0.10). The respective ovum recovery rates (M+/-SEM) were 55+/-9.8 and 74+/-13.2%. There was no effect of progesterone concentration in the priming phase on either the stages of development of the recovered ova or on their ability to develop during in vitro culture. It was concluded, therefore, that progesterone concentrations within the range 3.3 +/- 0.1 to 7.3 +/- 0.3 ng/ml during the priming phase and 2.4 +/- 0.3 to 6.5 +/- 0.2 ng/ml at the time of PMSG administration did not affect the ovulation rate or the viability of ova recovered from superovulated ewes.     

Comparison between PMSG- and FSH-induced superovulation for the generation of transgenic rats

Superovulation protocols using single injections of pregnant mare's serum gonadotropin (PMSG) or minipumps with follicle-stimulating hormone (FSH) were compared in immature Sprague-Dawley (SD) rats. We used the following criteria: total number of ova, rate of fertilization, in vitro embryo development, sensitivity of zygotes to the microinjection of foreign DNA into the pronucleus, and their in-vivo development after transplantation into the oviduct of a recipient. Female SD rats were stimulated with 15 IU PMSG or 10 mg FSH followed by the injection of human chorionic gonadotropin (hCG) at doses of 20 and 30 IU per female. After hCG administration, they were mated with males of the same strain and sacrificed on day 1 of pregnancy. The percentage of mated animals and the fertilization rate was similar in all groups. In rats given PMSG, the number of ovulated zygotes was hCG dose-dependent. In contrast, the dose of hCG did not influence the efficiency of superovulation in rats given FSH, which was equal to PMSG-treated rats at the optimal dose of hCG. The rates of in vitro blastocyst development (31.4 and 23.3%) and the resistance to microinjection into the pronucleus did also not differ significantly between zygotes of both studied groups. The proportion of offspring developing from microinjected zygotes after oviduct transfer (26.2 and 26.8%, respectively) and the rate of transgene integration per newborns (7.3 and 4.9%, respectively) was similar in both experimental groups. The results of this study demonstrate that superovulation of immature SD rats by PMSG is equally effective as FSH treatment and, thus, preferable for transgenic rat technology due to the lower costs and easier handling.     

Superovulation, collection and transfer of embryos and demi-embryos from Boran(Bos indicus ) cows and heifers

Twenty-three Boran(Bos indicus ) cows and heifers were superovulated with pregnant mare serum gonadotropin (PMSG); a total of four embryos and 4.1 +/- 0.3 (mean +/- SEM) ova per ova-producing donor resulted. Follicle stimulating hormone (FSH-P) was then used to superovulate 49 Boran cows for a total of 106 superovulations, of which 63 (59.4%) produced an average of 3.7 +/- 0.4 (mean +/- SEM) embryos. The embryo production was not influenced by either the season or the number of times(one to five) the cows were superovulated. A higher pregnancy rate was obtained when the selection of Boran recipients was based on their plasma-progesterone values (overall 52.5%, single embryos 63.3%, twin demi-embryos 45.8%) than when they were selected by palpation per rectum only (overall 43.8%, single embryos 50%, twin demi-embryos 36.4%). The twinning rate of twin demiembryos was 62.5%, whereas only single calves were born after transfer of two embryos per recipient. No pregnancies were produced following transfer of twin demi-embryos without zonae pellucidae. Transferring single demi-embryos gave a low pregnancy rate (13.3%). Twelve donor Boran cows (21 superovulations) bred with their fathers resulted in a high rate of early embryonic death; additionally, only 20.9% (overall) of the recipients became pregnant. Estrus synchronization of Boran cows with a progesterone releasing intravaginal device (PRID) for a short period (7 d) combined with one injection of prostaglandin (Day 6) produced a larger number of good quality recipients (70.5%) than using double prostaglandin injections (60%).