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.     

Induction of estrus in pubertal miniature gilts

Genetic engineering of miniature pigs has facilitated the development of numerous biomedical applications, such as xenotransplantation and animal models for human diseases. Manipulation of the estrus is one of the essential techniques for the generation of transgenic offspring. The purpose of the present study was to establish a useful method for induction of the estrus in miniature gilts. A total of 38 pubertal miniature gilts derived from 4 different strains were treated with exogenous gonadotropins. Estrus and ovulatory response were examined after treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as 200 IU PMSG and 100 IU hCG, 300 IU PMSG and 150 IU hCG, or 1,500 IU PMSG only, followed by 100, 150 or 750 IU hCG 72 h later, respectively. The optimal protocol was determined to be the combination treatment of 200 IU PMSG and 100 IU hCG followed by 100 IU hCG. The administration of 200 IU PMSG and 100 IU hCG was effective in inducing estrus regardless of the strain, although there was a strain difference in the ovulatory response. These results indicate that treatment with a low-dose combination of PMSG and hCG provides one of the simplest methods for induction of estrus and ovulation in pubertal miniature pigs.     

A comparison of 1- and 2-cell ova production by F2 50% Meishan versus F1 White line gilts

The objective of this study was to compare recovery of pronuclear and 2-cell ova from F2 50% Meishan (MX) gilts versus F1 White line (L42) gilts. Sexually mature MX and L42 gilts were allocated across 2 treatments: Super (MX:n=9; L42:n=10) and Control (MX:n=6; L42:n=5) in a 2 x 2 factorial experiment. Allyl trenbolone (AT) was used to synchronize estrus in all gilts. Super gilts were given pregnant mare serum gonadotropin (PMSG: 1250 IU) at 24 h after AT withdrawal. Eighty-five hours after PMSG administration, all Super gilts received 750 IU of human chorionic gonadotropin (hCG). Super gilts which exhibited estrus within 24 h of hCG administration (MX-Super: n=6; L42-Super: n=5) and all Control gilts were bred naturally to Line 3 boars at 12 and 24 hours after the onset of estrus. Ova were recovered from Super gilts between 60 and 64 h after hCG and Control gilts at 48 h after the onset of estrus. All 1- and 2-cell ova were centrifuged at 15000 x g and observed using differential interference contrast microscopy. The mean ovulation rate was greater (P<0.05) for both MX-Super and L42-Super gilts in comparison to their respective Control groups. No differences were detected in the mean ovulation rate (P>0.38) or the mean number of 1- and 2-cell ova recovered (P>0.50) between MX-Super and L42-Super gilts. The proportion of 1- and 2-cell ova which exhibited visible pronuclei or nuclei was also similar among MX-SUPER and L42-SUPER gilts. This study demonstrates that MX gilts respond/perform comparably to L42 gilts with respect to estrus synchronization, superovulation, ova yield, and the ease of visibility of pronuclei or nuclei in the ova.     

Production of transgenic rats using young Sprague-Dawley females treated with PMSG and hCG.

The aim of this study was to examine the effects of gonadotrophin treatments on estrus synchronization and superovulation in young Sprague-Dawley (SD) rats that had not yet exhibited defined estrus cycles (5 to 7 weeks old), and to produce transgenic rats using these females as embryo donors and recipients. In Experiment 1, female rats were injected with PMSG and hCG (12.5, 25, 50 and 100 IU/kg each) and were mated with stud males. The reproductive performance of young rats were highest when PMSG and hCG at doses of 25 IU/kg each were injected (delivery rate 87.5%, nursing rate 92.9%). In Experiment 2, female rats were injected with PMSG and hCG (100, 150 and 300 IU/kg each) to induce superovulation. More eggs were recovered from the rats injected with PMSG and hCG at 150 and 300 IU/kg than from those treated with 100 IU/kg (33.4 and 41.3 vs. 13.3 eggs per female, respectively; p < 0.05). In Experiment 3, pronuclear-stage zygotes from 150 IU/kg PMSG/hCG-treated rats were used for microinjection of the fusion gene of bovine alpha S1-casein gene promoter and human growth hormone gene (2.8 kb), and the microinjected zygotes were transferred into the oviduct ampullae of the 25 IU/kg PMSG/hCG-treated rats. Seventeen transgenic rats were obtained from the 334 DNA-injected zygotes (5.1%). These results indicate that recipients and embryo donors for the production of transgenic rats can be prepared by the appropriate PMSG and hCG treatments of young SD rats, regardless of their estrus stages.     

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.     

The effects of administration of gonadotrophins and estrogens on prenatal survival in gilts

In gilts ovulation occurs over a 4 to 8-hour period, with 70% of the ova being shed over a relatively short span of time. These oocytes supposedly give rise to more developed embryos at Days 10 to 12 which advance the uterine environment and reduce survival rates of less developed embryos because of an asynchronous environment. The aim of this experiment was to reduce embryo mortality by influencing the duration and pattern of ovulation. Crossbred gilts (n = 98) were bred at their first observed estrus after being exposed to boars at 200 days of age. Estrus detection was carried out daily at 0000, 0800 and 1600 hours. All gilts were artifically inseminated with fresh semen, with a minimum of 2.7 billion spermatozoa, at both 16 and 32 hours after detection of estrus. Gilts were randomly assigned to one of the following treatments at detection of estrus: 1) 500 IU (2ml) chorionic gonadotrophin (hCG) injected intravenously at the onset of estrus (n = 22); 2) 16 mug (4 ml) gonadotrophin releasing hormone (GnRH) injected intravenously at the onset of estrus (n = 25); 3) 11.5 mug estrogen added to the semen at the time of AI (n = 25); 4) control, untreated gilts (n = 26). All gilts were slaughtered at Day 30 of gestation (Day 0 = day of detected estrus). The mean (+/-SEM) number of ovulations in pregnant gilts per treatment was 13.0 +/- 0.52, 12.6+/-0.51, 13.6+/-0.54 and 13.3+/-0.52, while the mean (+/-SEM) number of normal embryos per treatment was 10.3+/-0.67, 10.5+/-0.66, 10.3 +/- 0.69 and 10.5 +/- 0.67 for hCG, GnRH, estrogen and control groups, respectively, for an embryonic survival rate of 80 +/- 4.2%, 83 +/- 4.1%, 74 +/- 4.3% and 79+/-4.2% in pregnant gilts. If nonpregnant gilts are included, the embryonic survival rate for treatments 1 to 4 was 76+/-7.0%, 73+/-6.5%, 60+/-6.5%, and 64+/-6.4%, respectively. There was no significant difference between treatments for any of these variables. There was no evidence that administration of hCG, or GnRH at the onset of estrus, or the addition of estrogen to semen improved embryonic survival in gilts by Day 30 in this experiment.     

Effects of progesterone pretreatment on fertility of gilts mated at an induced pubertal estrus

The effects of progesterone (100 mg/d, im) on pubertal fertility were examined in 247 gilts over 3 experiments. In the first experiment, 128 gilts were exposed to progesterone for 0, 2, 4 or 8 d before receiving PMSG (750 IU) 1 d later. The number of large (>4mm) follicles or corpora lutea (CL) were determined on the day of PMSG injection, Day 0 (onset of estrus), Day 1 or Day 10 (n=8). In the second experiment, embryonic survival was observed in 68 gilts after induction of estrus with PG600 (400 IU PMSG, 200 IU hCG). Vehicle or progesterone was previously administered for 2 d to these gilts, and they were allowed 1, 2, or 3 d between the last progesterone injection and PG600. In Experiment 3, a field trial was conducted in which 51 gilts received vehicle or progesterone for 2 d, followed by a 3-d interval before injection of PG600 to induce estrus. The gilts were allowed to farrow. Treatment with progesterone 1 d before PMSG increased (P<0.05) the number and size of preovulatory follicles and increased (P<0.05) the number of corpora lutea. However, the percentage of gilts pregnant by Day 10, the number of embryos recovered per gilt and embryonic survival were reduced (P<0.05) with progesterone pretreatment. Utilizing a smaller dose of PMSG (750 vs 400 IU) with PG600 negated the effects of progesterone pretreatment on ovulation rate. When the interval between progesterone treatment and PG600 was lengthened to 3 d embryonic survival to Day 30 improved but was similar to that of the vehicle/PG600 treated gilts. Fertility, as defined as conception rate and litter size, was similar between gilts exposed to vehicle or progesterone. These results indicate that pretreatment with progesterone up to the day before PMSG might improve follicular development and ovulation rate at the pubertal estrus with a dose of 750 IU of PMSG but not with the 400 IU (PG600). Reducing the dose of PMSG to 400 IU and allowing for 3 d between progesterone and gonadotropin treatment reduced the incidence of uterine infections but resulted in a fertility rate similar to that of gilts receiving PG600 alone.     

Induction of ovulation in gilts with cloprostenol

Prepuberal gilts were treated with 750 IU pregnant mare serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. In this model, ovulation occurred at 42 +/- 2 h post hCG treatment. When 500 mug of cloprostenol was injected at 34 and of 36 h after hCG injection, 78% of the preovulatory follicles ovulated by 38 h compared with 0% in the control gilts. In addition, plasma progesterone concentrations were significantly higher in the cloprostenol-treated group than in the control group (P<0.01) at 38 h, indicating luteinization along with premature ovulation. These results suggest that prostaglandin F(2)alpha (PGF(2)alpha) or an analog can be used to advance, synchronize or induce ovulation in gilts.     

Pituitary and ovarian hormone secretion and ovulation in gilts injected with gonadotropins during and after oral administration of progesterone agonist (Altrenogest)

We determined changes in plasma hormone concentrations in gilts after treatment with a progesterone agonist, Altrenogest (AT), and determined the effect of exogenous gonadotropins on ovulation and plasma hormone concentrations during AT treatment. Twenty-nine cyclic gilts were fed 20 mg of AT/(day X gilt) once daily for 15 days starting on Days 10 to 14 of their estrous cycle. The 16th day after starting AT was designated Day 1. In Experiment 1, the preovulatory luteinizing hormone (LH) surge occurred 5.6 days after cessation of AT feeding. Plasma follicle-stimulating hormone (FSH) increased simultaneously with the LH surge and then increased further to a maximum 2 to 3 days later. In Experiment 2, each of 23 gilts was assigned to one of the following treatment groups: 1) no additional AT or injections, n = 4; 2) no additional AT, 1200 IU of pregnant mare's serum gonadotropin (PMSG) on Day 1, n = 4); 3) AT continued through Day 10 and PMSG on Day 1, n = 5, 4) AT continued through Day 10, PMSG on Day 1, and 500 IU of human chorionic gonadotropin (hCG) on Day 5, n = 5; or 5) AT continued through Day 10 and no injections, n = 5. Gilts were bled once daily on Days 1-3 and 9-11, bled twice daily on Days 4-8, and killed on Day 11 to recover ovaries. Termination of AT feeding or injection of PMSG increased plasma estrogen and decreased plasma FSH between Day 1 and Day 4; plasma estrogen profiles did not differ significantly among groups after injection of PMSG (Groups 2-4). Feeding AT blocked estrus, the LH surge, and ovulation after injection of PMSG (Group 3); hCG on Day 5 following PMSG on Day 1 caused ovulation (Group 4). Although AT did not block the action of PMSG and hCG at the ovary, AT did block the mechanisms by which estrogen triggers the preovulatory LH surge and estrus.     

Synchronization of ovulation in cyclic gilts with porcine luteinizing hormone (pLH) and its effects on reproductive function

The overall objective was to evaluate the use of porcine luteinizing hormone (pLH) for synchronization of ovulation in cyclic gilts and its effect on reproductive function. In an initial study, four littermate pairs of cyclic gilts were given altrenogest (15 mg/d for 14 d). Gilts received 500 microg cloprostenol (Day 15), 600 IU equine chorionic gonadotropin (eCG) (Day 16) and either 5mg pLH or saline (Control) 80 h after eCG. Blood samples were collected every 4h, from 8h before pLH/saline treatment to the end of estrus. Following estrus detection, transcutaneous real-time ultrasonography and AI, all gilts were slaughtered 6d after the estimated time of ovulation. Peak plasma pLH concentrations (during the LH surge), as well as the amplitude of the LH surge, were greater in pLH-treated gilts than in the control (P=0.01). However, there were no significant differences between treatments in the timing and duration of estrus, or the timing of ovulation within the estrous period. In a second study, 45 cyclic gilts received altrenogest for 14-18d, 600 IU eCG (24h after last altrenogest), and 5mg pLH, 750 IU human chorionic gonadotropin (hCG), or saline, 80 h after eCG. For gilts given pLH or hCG, the diameter of the largest follicle before the onset of ovulation (mean+/-S.E.M.; 8.1+/-0.2 and 8.1+/-0.2mm, respectively) was smaller than in control gilts (8.6+/-0.2mm, P=0.05). The pLH and hCG groups ovulated sooner after treatment compared to the saline-treated group (43.2+/-2.5, 47.6+/-2.5 and 59.5+/-2.5h, respectively; P<0.01), with the most synchronous ovulation (P<0.01) in pLH-treated gilts. Embryo quality (total cell counts and embryo diameter) was not significantly different among groups. In conclusion, pLH reliably synchronized ovulation in cyclic gilts without significantly affecting embryo quality.     

Premature elevation of progesterone shortens duration of ovulation in PMSG/hCG-treated prepuberal gilts

A surge of LH during the follicular phase triggers multiple pathways, including progesterone and prostaglandin synthesis before culminating in ovulation. Progesterone has been shown to be involved in the ovulatory process in many species. In prepuberal gilts treated with PMSG/hCG the follicular progesterone level has been shown to increase sharply before ovulation. This study was conducted to investigate whether premature elevation of progesterone can accelerate the ovulatory process in Large White PMSG/hCG-treated prepuberal gilts. Fifty-four Large White gilts were treated with 1000 IU, i.m. PMSG to stimulate follicular growth, followed 72 h later by 500 IU, i.m. hCG to induce ovulation. Gilts in the treatment group (n = 27) were given progesterone intermuscularly at 24 and 36 h after hCG. Ovaries were exteriorized to observe ovulation points during laparotomy under general anesthesia at 38 to 50 h after hCG. Ovulation in both groups commenced by 40.05 h after hCG and was completed by 47.71 h in the control group and by 42.87 h after hCG in the treated group. Progesterone shortened (P < 0.01) ovulation time by 4.84 h and the time required (P < 0.01) for the median proportion of follicles to ovulate (40.7 vs 43.5 h after hCG). Progesterone also increased (P < 0.01) the plasma progesterone concentration without altering follicular progesterone concentration.     

Biochemical status of ovaries after induction of superovulation on different days of estrus cycle in mice

To investigate the role of ovarian status and to find out a suitable hormonal dose for induction of superovulation and its effect on biochemical status of the ovaries, the mice were injected with PMSG in doses of 5, 7.5, and 10 IU on different days of the estrous cycle i.e. proestrus, estrus, metestrus and diestrus followed by hCG injection 48 hr later. All these treatments increased the mean ovarian weight and ovulation rate when compared with that of control animals. Maximum response was observed by treatment with 7.5 IU PMSG on the day of estrus. This treatment resulted in a non-significant decrease in total proteins but a significant increase in the lipid concentrations while no change in cholesterol content of the ovaries of superovulated mice. The activity of acid phosphatase and lactate dehydrogenase significantly increased and alanine aminotranseferase significantly decreased in the ovaries of mice after superovulatory treatment when compared with that of control animals. This reveals that treatment with PMSG and hCG results in metabolic alterations in the ovaries which may perhaps be inducing biosynthetic deficiencies in oocytes as indicated by increased prenatal mortality in superovulated pregnant mice when compared with that of controls in the present studies.     

Influence of ovarian hyperstimulation and ovulation induction on the cytoskeletal dynamics and developmental competence of oocytes

This study was undertaken to determine the effects of gonadotrophin on cytoskeletal dynamics and embryo development and its role in improving the retrieval of developmentally competent oocytes. Female golden hamsters were injected with human chorionic gonadotrophin (hCG; 5-, 7.5- or 15-IU) on the day 4 of estrus, pregnant mare serum gonadotrophin (PMSG; 5-, 7.5- or 15-IU) on the day 1 of estrus, or 15-IU hCG at 56 hr post-15-IU PMSG injection in any cycle except estrus. Increasing the hCG dose decreased not only retrieval rate of 2-cell embryo but development to blastocyst after subsequent in vitro culture. Whereas, although increasing the PMSG dose induced increasing the number of 2-cell embryo and blastocyst, 15-IU PMSG injection caused retardation of development to blastocyst. No 2-cell embryos were retrieved by injecting both PMSG and hCG. The injections of 15-IU hCG and 7.5- or 15-IU PMSG inhibited the proliferation of trophectodermal and inner cell mass cells, respectively. Gonadotrophin injection didn't influence microtubular spindle formation, but 5- or 15-IU hCG, 15-IU PMSG, or PMSG and hCG injections induced aberrant cortical granule (CG) and microfilament distribution. After 15-IU hCG or PMSG and hCG injections, fewer oocytes had enriched cortical actin domains, and the expression of alpha-, beta- and gamma-actin genes was greatly increased. In conclusion, a high dose of gonadotrophins alters the microfilament and CG distribution, which in turn reduces the developmental competence of oocytes. Injecting a reduced dose of PMSG to initiate ovarian hyperstimulation without triggering ovulation contributes to the efficient retrieval of developmentally competent oocytes.     

In vitro development of embryos from superovulated gilts treated with the progesterone agonist, altrenogest (Regu-Mate) or the prostaglandin analogue, cloprostenol (Planate)

This study was conducted to compare the in vitro development of embryos from superovulated postpubertal gilts synchronized with progesterone agonist altrenogest (REG, Regu-Mate) and those from superovulated prepubertal gilts synchronized with prostaglandin analogue cloprostenol (PLA, Planate). Ten postpubertal gilts that had exhibited estrus at least once were fed 20 mg/d of REG from Day 0 (the first day of treatment, may have been any day of the estrous cycle) to Day 17. The gilts received intramuscularly (im) 1500 IU of equine chorionic gonadotropin (eCG) on the afternoon of Day 17, followed by 1000 IU of human chorionic gonadotropin (hCG) 84 h later. Eight prepubertal gilts received intramuscularly one dose of a combination of 400 IU of eCG and 200 IU of hCG (PG 600) on Day 0 (the first day of treatment), followed by 750 IU of hCG on Day 3. From Day 16 to Day 19, the prepubertal gilts received 350 mg/d of PLA, followed by 1500 IU of eCG on the afternoon of Day 19, then 1000 IU of hCG 84 h later. Gilts were checked for estrus with an intact boar. At estrus, all gilts were artificially inseminated and/or mated twice at 12-h intervals. Then 50 to 54 h after the hCG injection, a mid-ventral laparotomy was performed on each gilt. Corpora albicans (CA) and corpora hemorrhagica (CH) were counted, and oviducts were flushed in situ. The embryos recovered (1- to 2-cell) were cultured in modified Whitten's medium at 38.5 degrees C under an atmosphere of 5% CO2 in air for 144 h. The number of CA per gilt did not differ between the postpubertal and prepubertal gilts (11.9 vs 7.9, respectively; P > 0.05). However, the number of CH per gilt (27.5 vs 18.1, P = 0.05) and the number of embryos per gilt (26.2 vs 15.3, P < 0.05) were higher in postpubertal gilts than in prepubertal gilts. Furthermore, after 144 h of in vitro culture, the percentage of embryos cleaving to the >-16-cell (morula + blastocysts) or > or =32-cell (blastocysts) was greater (P < 0.05) in prepubertal gilts than in postpubertal gilts (85.2 vs 68.5, 55.7 vs 44.2, respectively). The total numbers of embryos examined were 122 and 260 in prepubertal and postpubertal gilts, respectively. These results show that postpubertal gilts treated with REG produced a higher number of embryos. However, better embryo development was noted with zygotes from prepubertal gilts primed with exogenous gonadotrophin, followed by synchronization with prostaglandin before induction of superovulation and insemination.     

Embryo production and endocrine response in ewes superovulated with PMSG, with or without monoclonal anti-PMSG administered at different times

Mature nonlactating Altamurana ewes (n = 168) were synchronized in the seasonal anestrus period with FGA-impregnated intravaginal pessaries for 12 d. In Experiment 1, 48 ewes were divided into a 3 x 4 factorial design for anti-PMSG monoclonal antibody (AP) bioassay test. Concomitant injections of PMSG (1000, 1500, 2000 IU) and AP (0, 1, 2, 3 microl/IU PMSG) were given, and ovarian response was evaluated by laparoscopy. In Experiment 2, 120 ewes were divided into 8 experimental groups (n = 15 per group). The ewes treated with 1000 or 1500 IU PMSG at -24 h from sponge removal were given AP intravenously at 50 h after pessary withdrawal, 12 or 24 h after the onset of estrus, while the controls did not receive AP. Blood samples were collected from ewes (n = 6) treated with 1500 IU PMSG with or without anti-PMSG. Ovarian response and embryo production were evaluated on Day 7 after sponge removal upon laparotomy. It was found that 1 microl AP was effective in neutralizing 1 IU PMSG. No significant differences in serum concentrations of progesterone were observed among the groups of superovulated ewes. Estradiol-17 beta levels were reduced following AP treatment 12 h after the onset of estrus. At a lower dosage of superovulatory treatment (1000 IU PMSG), AP injected at 12 or 24 h after the onset of estrus significantly lowered large follicles (P < 0.01) and increased the rate of ovulation (P < 0.05). Moreover, embryo production showed a more than two-fold increase (P < 0.01) of viable embryos following AP injection at 12 or 24 h after the onset of estrus (3.2 to 3.3 vs 1.3, with vs without anti-PMSG). It is concluded that superovulatory treatment with 1000 IU PMSG plus AP administered at a fixed time after the onset of estrus may improve ovarian response and the yield of viable embryos in ewes.     

Estrous cycle stage-independent treatment of PMSG and hCG can induce superovulation in adult Wistar-Imamichi rats.

The estrous cycle influence on the number of ovulated eggs after injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was investigated in 12, 18, and 24 weeks old adult female Wistar-Imamichi (WI) rats. PMSG (150 IU/kg) was injected at metestrus, diestrus, proestrus, or estrus, followed by hCG (75 IU/kg) 55 h later. Ovulation was induced at all ages and stages of the estrous cycle. The number of ovulated eggs was not affected by stage for similarly aged rats, however, the number of ovulated eggs obtained after treatment decreased with age. These results demonstrate that the PMSG/hCG treatment can induce ovulation at any stage of estrous cycle in WI rats and efficient superovulation at 12 weeks of age.     

Estrus, ovulation and conception following timed insemination in Romney ewes treated with progestagen and gonadotropins.

The estrus — ovulation time relationships was examined in Romney ewes treated with progestogen (intravaginal sponge) and gonadotropins (PMSG + HCG or PMSG alone) prior to (January) and during (April) the breeding season. The conception rate of ewes inseminated at predetermined times after treatment was also investigated.Ewes exhibited estrus sooner after sponge removal in April than in January (34.9 v 38.9 hrs, P < 0.001). The interval from sponge removal to ovulation was also shorter in April than in January (56.3 – 62.1 hrs, P < 0.01). There were no significant differences between treatments or season on the mean interval from estrus to ovulation. Types of gonadotropin treatment had no effect on the estrus — ovulation time relationships. There were no significant effects of season, hormone treatment or time of insemination on lambing rate.     

Reversal of indomethacin blockade of ovulation in gilts by prostaglandins

Prepubertal gilts given 750 IU pregnant mares′ serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation fail to ovulate when 10 mg/kg indomethacin (INDO) is injected 24 h after hCG administration. This study examines the effects of administration of exogenous prostaglandins F_2α and E₂ (PGF_2α and PGE₂) alone or in combination, and at various times prior to the expected time of ovulation, on the INDO blockade of ovulation in PMSG/hCG-treated gilts. Occurrence of ovulation was determined by visual observation at laparotomy 48 h after hCG. When 5 mg or 10 mg PGF_2α was injected at each of 38, 40 and 42 h after hCG injection, 63% and 79%, respectively, of preovulatory follicles ovulated. In contrast, injection of 5 mg PGE₂ or 5 mg PGE₂ plus 5 mg PGF_2α induced ovulation in 0% and 24% of preovulatory follicles, respectively. In control groups, 100% of folicles in PMSG/hCG-treated gilts ovulated whereas none did so in PMSG/hCG/INDO-treated animals. These results indicate that administration of PGF_2α can induce ovulation in the PMSG/hCG/INDO-treated prepubertal gilt and suggest that PGE₂ is ineffective and may be antagonistic to PGF_2α in overcoming the ovulation blocking effect of INDO.     

Cytogenetic analysis of Day-4 embryos from PMSG/hCG-treated prepuberal gilts

Prepuberal gilts were treated with pregnant mare serum gonadotropin (PMSG) to study the effects of its dosage on ovulation rate, fertilization rate after artificial insemination, embryo viability, and rate of development and incidence of chromosome abnormalities in Day-4 embryos. Gilts received 750 IU, 1250 IU or 1500 IU of PMSG, followed 72 h later by 500 IU human chorionic gonadotropin (hCG). Gilts were inseminated 28 to 30 h following the hCG injection, and resulting embryos were collected on Day 4 post ovulation. Ovulation rate was higher in the 1250 IU group than in the 1500 IU group or the 750 IU group. The 1500 IU dose caused excessive stimulation of the ovary, resulting in the occurrence of large (>10mm diameter) unovulated follicles, reduced fertilization rate and low embryo recovery rate. There was no difference in the incidence of chromosome abnormalities among the three groups, although the 1500 IU group had higher embryonic mortality than the two lower dose groups. A dose of 1250 IU PMSG increased ovulation rate above that achieved by 750 IU and, therefore, increased the number of oocytes or embryos available for transfer or for other studies, without sacrificing embryo viability or increasing the incidence of chromosome abnormalities.