In vitro production of bovine embryos: developmental competence is acquired before maturation

Few studies have examined the importance of the time during which oocytes are left in the ovaries following animal slaughter. The objective of this study was to determine the optimal time for retrieving oocytes after slaughter and to ascertain if superovulating cows in association with this optimal time could increase the developmental competence of bovine oocytes. In Experiment 1, oocytes were left in the postmortem ovaries for 2,3,4,5,6 or 7 h and were then transported to the laboratory at approximately 30 degrees C. Recovered oocytes were processed in vitro using standard techniques. In Experiment 2, cyclic heifers (n = 18) were superovulated between Days 8 and 12 of the estrous cycle with 8 constant doses (4 mg each, twice daily) or 8 decreasing doses (2 injections of 4,3,2 and 1 mg every 12 h) of FSH-P +/- 1 mg prostaglandin 24 or 48 h before slaughter. Oocytes were left in the ovaries for 4 h and were classified according to the state of their cumulus and cytoplasm. The results indicated that oocytes aspirated from ovaries collected 4 h after slaughter produced significantly more > or =64-cell embryos after 7 d of in vitro development than those collected 2, 6 or 7 h postslaughter. Oocytes (87%) from superovulated animals had numerous layers of cumulus cells and originated from medium (2.7 to 8 mm) and large (> or =8 mm) follicles. Significantly more oocytes developed from large follicles than from medium follicles. Although individual culture of the oocytes negatively affected the percentage of embryos produced, group culture of oocytes from animals that were superovulated and left in the postmortem ovaries for 4 h resulted in exceptionally high rates of embryos after 5 d of IVD. On average, 60 to 80% of 16-cell embryos were produced, indicating that under the proper conditions, developmental competence is acquired before in vitro maturation.     

Timing of the onset and duration of ovulation in superovulated beef heifers

Thirty-two beef heifers were induced to superovulate by the administration of follicle stimulating hormone-porcine (FSH-P). All heifers received 32 mg FSH-P (total dose) which was injected twice daily in decreasing amounts for 4 d commencing on Days 8 to 10 of the estrous cycle. Cloprostenol was administered at 60 and 72 h after the first injection of FSH-P. Heifers were observed for estrus every 6 h and were slaughtered at known times between 48 to 100 h after the first cloprostenol treatment. The populations of ovulated and nonovulated follicles in the ovaries were quantified immediately after slaughter. Blood samples were taken at 2-h intervals from six heifers from 24 h after cloprostenol treatment until slaughter and the plasma was assayed for luteinizing hormone (LH) concentrations. The interval from cloprostenol injection to the onset of estrus was 41.3 +/- 1.25 h (n = 20). The interval from cloprostenol injection to the preovulatory peak of LH was 43.3 +/- 1.69 h (n = 6). No ovulations were observed in animals slaughtered prior to 64.5 h after cloprostenol (n = 12). After 64.5 h, ovulation had commenced in all animals except in one animal slaughtered at 65.5 h. The ovulation rate varied from 4 to 50 ovulations. Approximately 80% of large follicles (> 10 mm diameter) had ovulated within 12 h of the onset of ovulation. Onset of ovulation was followed by a dramatic decrease in the number of large follicles (> 10 mm) and an increase in the number of small follicles (相似文献   

Superovulation can reduce the developmental competence of bovine embryos

This study was done to determine if different superovulatory regimens could have an effect on the percentage of embryos produced using IVM/IVF/IVC. Cyclic heifers (n = 22) were superovulated between Days 8 and 12 of the estrous cycle with 4, 6 or 8 constant doses of FSH-P (4 mg each, twice daily) +/- the addition of 1 mg prostaglandin 24 h before slaughter. Ovaries from these superovulated cows and from untreated cows were collected and the follicles dissected. Oocytes were classified according to the appearance of their cumulus and cytoplasm. Individual culture as well as group culture were performed but an individual culture reduced the percentage of oocytes developing into embryos for both untreated and superovulated animals. The results indicated that despite the superovulation regimen the developmental competence of the oocytes collected was lower (0 to 15% embryos) than that of oocytes from untreated animals (20 to 34% embryos). Small follicles ( < or = 2.7 mm) yielded mostly oocytes with an incomplete or partially expanded cumulus investment that never developed into an embryo. Differences in the morphology of the oocytes from medium (2.7 to 8 mm) and large ( > or = 8 mm) follicles were apparent, but equal developmental rates were obtained between all classes of oocytes (12 and 8% embryos, respectively). Follicular atresia was reduced significantly after superovulation (81% nonatretic follicles in treated vs 42% nonatretic follicles in untreated animals); however oocytes from atretic and slightly atretic follicles developed similarly to those from nonatretic follicles. These results suggest that although superovulation increases follicular size and decreases atresia, these conditions are not sufficient to confer developmental competence on the oocytes.     

Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovulation and follicular growth in gonadotropin-treated gilts followed by in vitro fertilization and development of their oocytes

We investigated whether porcine ovaries derived from FSH-pituitary (FSH-P) or hCG-treated animals can produce oocytes with better in vitro cytoplasmic maturation and in vitro embryonic development relative to those derived from saline-treated animals. The size of the follicle producing the oocyte was also studied. Each of 25 prepubertal gilts received 1 of 6 treatments by intramuscular injection: 1) saline (3 mL, once, n = 5); 2) FSH-P8-3 (8 mg, 3 times, with a 24-h interval, n = 4); 3) FSH-P16-3 (16 mg, 3 times, with a 24-h interval, n = 4); 4) FSH-P16-1-P4-2 (16 mg, once, 4 mg, twice, with a 24-h interval, n = 4); 5) FSH-P16-1 (16 mg, once, n = 4); or 6) hCG (100 IU, 3 times, with a 24-h interval, n = 4). The ovaries were removed by mid-ventral laparotomy 72 h after the first injection. The numbers of corpora hemorrhagica (CH) with each FSH-P treatment were similar (P > 0.05). However, compared with gilts treated with saline or hCG, those treated with FSH-P8-3 had a greater (P < 0.05) number of CH. Treatment with FSH-P8-3 or FSH-P16-3 induced significant growth of medium/large follicles (4 to 8 mm in diameter) compared with saline or FSH-P16-1. The same results were observed when FSH-P8-3 was compared with FSH-P16-P4-2 or hCG. After in vitro fertilization, the rates of male and female pronuclei in oocytes derived from medium/large follicles did not differ (P > 0.05) between treatments, but in oocytes derived from small follicles they were lower (P < 0.05) in saline-treated than in FSH-P16-1-P4-2-treated gilts. After 120 h in culture, the percentages of the inseminated oocytes from 1 to 3 mm or 4 to 8 mm follicles developing to > or = 2-cell did not differ (P > 0.05) between saline- and gonadotropin-treated gilts. However, a higher (P < 0.05) percentage of the inseminated oocytes from 4 to 8 mm follicles had developed to the morula stage or beyond, than those from the 1 to 3 mm follicles. In conclusion, administration of single or multiple doses of FSH-P induced ovulation, but only 8 or 16 mg FSH-P injected 3 times with 24-h intervals for 72 h induced growth of 4 to 8 mm follicles. The size of follicle from which the oocyte derived also had a significant effect on its development in vitro.     

The dominant follicle exerts an interovarian inhibition on FSH-induced follicular development

An experiment was conducted to evaluate the role of the dominant follicle (DF) of the first wave in regulating follicular and ovulatory responses and embryonic yield to a superovulation regime with FSH-P. Twenty normally cycling Holstein-Freisian heifers (n = 20) were synchronized with GnRH and pgf(2alpha) and randomly assigned to a control or a treated group (n = 10 each). Treated heifers had the first wave dominant follicle removed via transvaginal, ultrasound-guided aspiration on Day 6 after a synchronized estrus. All heifers received a total of 32 mg FSH-P given in decreasing doses at 12 h intervals from Day 8 to Day 11 plus two injections of pgf(2alpha) (35 mg and 20 mg, respectively) on Day 10. Heifers were inseminated at 6 h and 16 h after onset of estrus. Follicular dynamics were examined daily by transrectal ultrasonography from Day 4 to estrus, once following ovulation, and at the time of embryo collection on Day 7. Blood samples were collected daily during the superovulatory treatment and at embryo collection. Follicles were classified as: small, /= 10 mm. Aspiration of the dominant follicle was associated with an immediate decrease in large follicles, and a linear rate increase in small follicles from Day 4 to Day 8 just prior to the FSH-P injections, (treatment > control: +0.33 vs. -0.22, number of small follicles per day; P < 0.10). During FSH-P injections, the increase in number of medium follicles was greater (P < 0.01) for treatment on Day 9-11 (treatment > control: Day 9, 3.2 > 1.8; Day 10, 9.2 > 4.7; Day 11, 13.1 > 8.3; +/- 0.56). Number of large follicles was greater in treatment at Day 11 (5.12 > 1.4 +/-0.21; P < 0.01). Mean number of induced ovulatory follicles (difference between number of follicles at estrus and Day 2 after estrus) was greater in treatment (13.4 > 6.3 +/- 1.82; P < 0.01). Plasma estradiol at Day 11 during FSH-P treatment was greater in treatment (32.5 > 15.8 +/- 2.6; P < 0.01). Plasma progesterone at embryo flushing (Day 7 after ovulation) was greater in treatment (7.4 > 4.9; P < 0.02); technical difficulties at embryo recovery reduced sensitivity of embryonic measurements. No changes in the distribution of unfertilized oocytes and embryo developmental stages were detected between control and treatment groups. Presence of dominant follicle of the first wave inhibited intraovarian follicular responses to exogenous FSH.     

The time interval between FSH administration and ovarian aspiration influences the development of cattle oocytes

Depriving the ovary of exogenous FSH for 1, 2 or 3 d following a bolus injection of FSH was shown to influence the quality of the recovered oocytes. Thus, we compared the developmental competence of oocytes from heifers which had been stimulated for 3 d with FSH (Folltropin-V) and, after an interval of 36, 48 or 60 h, underwent blind transvaginal aspiration. The ovaries of heifers with a palpable or functional corpus luteum were aspirated to remove all large follicles 2 d prior to being injected with either 6 doses of saline (S), 6 doses (20 mg/mL) of FSH (F), or in 6 decreasing doses of FSH (3, 3, 2, 2, 1, 1 mL; Fd). Follicles were counted and classified (medium: 5 to 10 mm, large: >10 mm) with ultrasonography before each aspiration. The oocytes recovered were classified, matured, fertilized, and developed in vitro. On a per animal basis, 1.5, 5.2 and 4.7 large and 1.5, 10.7 and 10.7 medium follicles were counted for S, F and Fd, respectively. A mean of 3.3, 9.1 and 7.7 oocytes was recovered for treatments S, F and Fd, respectively and 58, 94 and 82% were enclosed in a nonexpanded cumulus or a corona layer. Oocyte development rates were based on counts of embryos with 32 or more nuclei at Day 6.5. When oocytes were recovered 36 h after the last injection, an average of 1, 2.7 and 2 embryos per animal was obtained with S, F and Fd, respectively; at 48 h, 0.75, 4.25 and 1 embryo; and at 60 h, 0, 2.5 and 2.7 embryos. Variance analysis was performed, and the protected LSD test indicated that treatment F at 48 h resulted in a significantly higher embryo rate than Fd at 48 h (P<0.05) or S (all times; P<0.05). The reduced effect of the Fd regimen could be due to the decreasing FSH support during follicular growth or to the lower total amount of FSH given. In conclusion, these results indicate an advantage of using moderate (3 d) follicle stimulation followed by a period of FSH starvation to obtain optimal embryo production.     

Superovulation of beef heifers with Pergonal (HMG): A dose response trial

A study was designed to establish a dose-response curve for Pergonal (Human Menopausal Gonadotrophin) and to compare its efficacy in inducing superovulation with commercial FSH-P. A recognized treatment schedule for HMG of two ampoules at 0, 12, 24 and 36 hours and one ampoule at 48, 60, 72, 84, 96 and 108 hours was considered to be our 100% effective dose level. Fifty mature cycling cross-bred beef heifers were superovulated on day 10 +/- 1 of their cycle. Treatment groups were HMG I (200% dose), HMG II (100% dose), HMG III )50% dose), HMG IV (25% dose) and FSH-P (total dose 32 mg). All groups received 500 ug of cloprostenol 72 hours after initiation of treatments. The heifers were observed for onset of estrus and inseminated at 12, 24 and 36 hours. All heifers were slaughtered on day 7 post-estrus and their reproductive tracts removed for processing. All heifers were bled once daily for progesterone estimation and four times daily for two days beginning 24 hours after cloprostenol injection, for luteinizing hormone and estradiol-17beta estimations. A dose response to HMG was demonstrated for number of corpora lutea and all classes of ova/embryos. HMG II (100% dose) closely approximated the optimum dose for superovulation. There was no significant difference between the HMG II group and the FSH-P group for mean number of transferable embryos. The 200% HMG dose did not increase the numbers of ovulations or ova recovered but did decrease the numbers of fertilized and transferable ova.     

Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization

The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos.     

Effects of FSH commercial preparation and follicular status on follicular growth and superovulatory response in Spanish Merino ewes

Ovarian follicular development was characterized in 24 Spanish Merino ewes to study effects of the follicular status and the FSH commercial product used on follicular growth and subsequent superovulatory response. Estrus was synchronized using 40 mg fluorogestone acetate sponges. The superovulatory treatment consisted in 2 daily i.m. injections of FSH from 48 h before to 12 h after sponge removal. Sheep were assigned randomly to 2 groups treated with 6 decreasing doses (4, 4, 3, 3, 2, 2 mg) of FSH-P or with 6 doses of 1.25 mL of OVAGEN. Growth and regression of all follicles > or = 2 mm were observed by transrectal ultrasonography, and recorded daily from Day 6 before sponge insertion to the first FSH injection, and then twice daily until estrus was detected with vasectomized rams. Differences were detected in follicular development from the first FSH injection to detection of estrus (-48 to 36 h from sponge removal) between groups. Administration of FSH-P increased the appearance of new follicles with respect to OVAGEN (6.3 +/- 0.7 vs 4.8 +/- 0.4; P < 0.05), and the mean number of medium (4 to 5 mm) follicles (8.9 +/- 1.2 vs 6.6 +/- 0.9; P < 0.05). However, the mean number of follicles that regressed in size after sponge removal (5.9 +/- 0.4 vs 3.3 +/- 0.4) and the number of preovulatory sized follicles that did not ovulate (60 vs 42.4%) were also higher in FSH-P treated ewes (P < 0.05). So, finally, there were no differences in ovulation rate, as determined by laparoscopy on Day 7 after sponge removal, between ewes treated with FSH-P or OVAGEN (6.3 +/- 1.9 vs 7.0 +/- 1.7 CL). In all the ewes, the ovulatory response was related (P < 0.05) both to the number of small follicles (2 to 3 mm in diameter) present in the ovaries at the start of treatment with exogenous FSH and to the number of follicles that reached > or = 4 mm in size at estrus, despite differences in the pattern of follicular development when using different commercial products.     

Effects of passive immunization against inhibin on ovulation rate and embryo recovery in holstein heifers

The effects of acute neutralization of endogenous inhibin on ovulation rate and circulating FSH levels were investigated. Nine or ten days after estrus, 5 heifers were given a single injection of 75 ml iv inhibin antiserum produced in a castrated male goat, while another 5 were given the same amount of a castrated male goat serum. All heifers were given injections of PGF2alpha im at 48 h and 60 h after the serum injection. Those exhibiting an estrus were artificially inseminated with frozen-thawed semen. Seven or eight days after the insemination, ova or embryos were collected using a non-surgical method. Administration of inhibin antiserum resulted in a significant increase in the number of medium-sized follicles compared with the number in the control animals. The number of large follicles in the inhibin-neutralized animals was 4.8 +/- 2.4 (mean +/- SEM; n = 5) on the day of estrus, while there was a single large follicles in the ovaries of control animals. Seven or eight days after estrus, 3 to 16 ova or embryos were recovered from 4 of 5 animals, and 64 % of the total ova/embryos were transferable. Administration of inhibin antiserum produced a significant increase in the concentrations of plasma FSH from 12 to 72 h after the serum injection compared with the levels in the control animals (P < 0.05). After the onset of estrus, preovulatory LH and FSH surges were noted in inhibin-neutralized animals and magnitude of the rise in each hormone was similar to the control animals. The present study demonstrates that a single injection of the inhibin antiserum induces multiple ovulations probably by enhancing FSH secretion, and that recovery of embryos is equal to that observation after an ordinary FSH treatment.     

In vitro techniques of bovine oocyte maturation, fertilization and embryo culture resulting in the birth of a calf

Oocyte cumulus complexes were aspirated from 3 to 5 mm follicles of cows prestimulated with 2.000 IU PMSG 24 h before slaughter. Oocytes matured in culture were fertilized in vitro by heparinized freshly ejaculated or epididymal spermatozoa. The cultivation procedure for fertilized eggs was the same as that used for cultivation of oocytes. From 163 matured oocytes, 109 cleaved to the 2-cell stage 24 h after fertilization and after 6 days of cultivation, 18 developed to the late morula and 18 to the blastocyst stages. Eleven blastocyts and 1 late morula were transferred surgically to the uteri of 7 recipient heifers. Two heifers became pregnant: one delivered a bull-calf at term, while the other pregnancy resulted in abortion at the 3rd month. The examination of some embryos by transmission electron microscopy showed an almost normal morphology for most cells. The degenerated cells contained mostly electron-dense residual bodies of unknown origin.     

Cytogenetic analysis of caprine 2- to 4-cell embryos produced in vitro

Prepubertal goat in vitro matured/in vitro fertilised oocytes produce only a small percentage of blastocysts. The present study examines the incidence of chromosomal anomalies in 2- to 4-cell embryos in vitro produced (IVP) from prepubertal oocytes fertilised with the semen of two males. Cumulus-oocyte complexes were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM199 supplemented with 20% heat inactivated Donor Bovine Serum (DBS), 10 microg/ml FSH + 10 microg/ml LH + 1 microg/ml 17beta-oestradiol for 27 h at 38.5 degrees C in 5% CO2 in air. IVM oocytes were inseminated with the sperm from two males prepared using the swim-up and heparin-capacitation procedures. At 24 h postinsemination (hpi) the oocytes were transferred to 100 microl drops of SOF medium for a further 24 h. At 17 hpi a sample of oocytes was stained with lacmoid to evaluate the nuclear stage after fertilisation. The cleavage rate was determined at 24, 36 and 48 hpi and chromosome slides were prepared according to the gradual-fixation technique and stained with Leishman. A total of 1070 2- to 4-cell embryos from prepubertal goat oocytes were studied, but it was only possible to analyse 241 cytogenetically. Of these, 40% exhibited a normal diploid chromosome complement, 59% were haploid and 1% were triploid. There were significant differences between the two males in sperm oocyte penetration and oocyte cleavage but no differences were found in chromosomal anomalies. In conclusion, the low number of embryos karyotyped and the high number of haploid embryos found in this study suggested a high incidence of abnormal fertilised embryos and deficient cytoplasmic maturation of the oocyte which inhibits sperm head decondensation.     

Effects of gonadotropin treatment on ovarian follicle growth, oocyte quality and in vitro fertilization of oocytes in prepubertal gilts

This study was undertaken to compare the effects of FSH-pituitary (FSH-P), eCG, and a combination of gonadotropins containing 400 IU eCG and 200 IU hCG (PG 600) on the growth of large follicles, oocyte quality and in vitro fertilization (IVF) rate of in vitro matured (IVM) oocytes in prepubertal gilts. The ovaries were removed via midventral laparotomy 48 h (Experiment 1) or 72 h (Experiment 2) after the first injection. In Experiment 1, 30 gilts received 1 of 5 treatments: 1) saline (3 ml i.m., once, n = 6); 2) FSH-P8 (8 mg i.m., twice, with a 24-h interval, n = 6); 3) FSH-P16 (16 mg i.m., twice, with a 24-h interval, n = 6; 4) eCG (1000 IU i.m., once, n = 6); or 5) PG 600 (5 ml i.m., once, n = 6). Compared with saline, treatment with PG 600 or eCG induced significant (P < 0.05) growth of large follicles (> or = 6 mm). In Experiment 2, 16 gilts received 1 of 5 treatments: 1) saline (n = 4); 2) FSH-P8 (n = 4); 3) FSH-P16 (n = 4); 4) eCG (n = 4), or 5) PG 600 (n = 4). The same injection protocol as in Experiment 1 was used. Compared with treatment with FSH-P8 or FSH-P16, eCG increased (P<0.05) the number of large follicles. The proportion of good oocytes was increased (P<0.05) with FSH-P8 or FSH-P16 compared with treatment with eCG or PG 600. Moreover, oocytes from eCG-treated gilts had a greater (P<0.05) rate of male and female pronuclei than FSH-P or saline-treated gilts. In conclusion, treatment with FSH-P resulted in a higher proportion of oocytes with multilayer cumulus cells, whereas treatment with eCG resulted in higher pronuclear rates following in vitro fertilization in prepubertal gilts.     

Plasma concentrations of luteinizing hormone and progesterone during superovulation of dairy cows using follicle stimulating hormone or pregnant mare serum gonadotrophin

Eighteen lactating Holstein cows were randomly divided into three groups of equal size. Six cows were not superovulated; the remaining cows were superovulated using either FSH-P or PMSG beginning on Day 12 of the estrous cycle (day of ovulation = Day 0). Animals treated with FSH-P were injected intramuscularly (i.m.) with 4 mg FSH-P every 12 h for 5 d. PMSG was administered i.m. as a single injection of 2350 IU. Cloprostenol (PG, 500 ug) was injected i.m. 56 and 72 h after commencement of treatment and at the same time in the cycle of controls. All cows were inseminated 56, 68 and 80 h after the first PG injection. Blood samples (5 ml) were collected daily and every 15 min for a period of 9 h on Days -1, 0, 2, 8 and 10, with continuous blood sampling at 15-min intervals during Days 3 to 6. Ovulation rate was 27.7 +/- 8.22 in animals treated with PMSG, and 8.0 +/- 3.2 embryos per donor were recovered. In the FSH group, ovulation rate was 8.3 +/- 1.48 and 3.0 +/- 1.1 embryos per donor were recovered. Progesterone concentrations were similar in all three groups until the onset of the LH surge, when progesterone concentrations were greater (P<0.05) in animals of the PMSG group. After the preovulatory LH surge, concentrations of progesterone started increasing earlier (44 h) in cows treated with PMSG, followed by FSH-treated cows (76 h) and controls (99 h). The LH surge occurred earlier (P<0.05) in PMSG-treated cows (37 h after first PG treatment), than in animals treated with FSH-P (52 h) or controls (82 h). In animals treated with FSH-P, the magnitude of the preovulatory LH surge (24.2 +/- 1.02 ng/ml) was higher (P<0.05) than in the other two groups (PMSG = 17.1 +/- 2.04 ng/ml; control, 16.7 +/- 1.24 ng/ml). Superovulation with FSH-P or PMSG did not affect either mean basal LH concentration, frequency or amplitude of LH pulses during Days -1, 0, 2, 3, presurge periods, or Days 8 and 10 post-treatment. At ovariectomy, 8 d post-estrus, more follicles > 10 mm diam. were observed in the ovaries after treatment with PMSG (8.5 +/- 5.66) than after treatment with FSH-P (0.7 +/- 0.42) (P<0.05). Maximum concentrations of PMSG were measured 24 h after administration. Following this peak, PMSG levels declined with two slopes, with half-lives of 36 h and 370 h.     

Interspecific transfer of IVM IVF-derived red sheep (Ovis orientalis gmelini ) embryos to domestic sheep (Ovis aries )

Two embryo production methodologies were investigated to generate Red sheep embryos for use in an interspecific embryo transfer program. In Experiment 1, 4 multiparous female Red sheep (Ovis orientalis gmelini ) were implanted with CIDR type G devices for 11 d. Forty-eight hours prior to CIDR removal, a total of 22.5 mg bid of FSH-P was administered over a 3-d period. Laparoscopic embryo collection was performed 5 d post breeding, and embryos were transferred to domestic recipient ewes (Ovis aries and Ovis orientalis musimon ). In Experiment 2, 7 nulliparous female Red sheep were implanted with CIDR devices and injected with 200 IU of PMSG and 25 mg of FSH-P on the 8th day of implant insertion. At 60 to 70 h post PMSG/FSH-P treatment, follicular oocytes were aspirated laparoscopically. The recovered oocytes were matured in M199 (with fetal calf serum, FSH, LH, penicillin and streptomycin) at 39 degrees C in a humidified atmosphere containing 5% CO(2). At 24 h oocytes were fertilized with frozen-thawed semen at a concentration of 1.6 x 10(6) sperm/ml. The ova/embryos were placed in CR2 or BOEC culture medium at 20-22 h post IVF. Following 3 to 4 d in culture, embryos were transferred laparoscopically to the uterine horn of synchronized recipients. In Experiment 1, 4 embryos and 6 UFO were collected from 2 embryo donors, respectively. Two embryos were transferred with the aid of a laparoscope to each of 2 Rambouillet recipients, one of which gave birth to a healthy Red sheep lamb at 158 d of gestation. In Experiment2, a total of 62 oocytes was collected from 7 oocyte donors; 16 developed to the 16- to 32-cell stage and were transferred to 8 recipients. Three of these IVM-IVF embryos were transferred laparoscopically to 2 Mouflon recipients, resulting in no pregnancies. Thirteen IVM-IVF embryos were transferred to 6 Rambouillet recipients. Each of these gave birth to a single healthy Red sheep lamb. Gestation lengths of the 3 IVM-IVF lambs ranged from 152 to 162 d. This research demonstrates that when using compatible species IVM-IVF technology in conjunction with interspecific ET can lead to the production of live offspring and can be used to propagate exotic ovine species.     

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 3. In vitro fertilisation using sheep serum as a capacitating agent and the subsequent birth of calves

The following experiments investigated the use of sheep serum (SS) as a capacitating agent for red deer (Cervus elaphus) sperm during in vitro fertilisation. Red deer oocytes were collected at slaughter and matured in vitro for 24h in TCM-199 supplemented with 10% foetal calf serum, 10 microg ml(-1) FSH and LH, and 1microg ml(-1) of oestradiol. Fertilisation medium was IVF-SOF modified to contain 5mM Ca(2+) and no glucose. Experiment 1 investigated the addition of heparin, BSA (8 mg ml(-1)) or 20% SS. All oocytes were penetrated when IVF-SOF was supplemented with SS compared to 10 and 0% penetration when either heparin or BSA was present (P<0.01). However, 43.8% of these oocytes were polyspermic when the medium contained SS. In Experiment 2, the effect of sperm concentration on penetration rates during in vitro fertilisation was investigated. Total sperm penetration and monospermic penetration rates increased with increased sperm concentrations in a log linear manner (P<0.001) and both approached an asymptote at 0.4 x 10(6) sperm ml(-1) with 93.6 and 77% for total and monospermic penetration, respectively. Polyspermic fertilisation also increased with increasing sperm concentrations (P<0.05) but was variable (range 3.5+/-4.2 to 42.3+/-10.6%), especially at the lower sperm concentrations. Experiment 3 investigated the viability of these oocytes after transfer into red deer recipients. Fifteen 2- and 4-cell embryos were transferred into the oviducts of synchronized recipients 28 h post in vitro insemination. An additional fourteen embryos (8-10 cell) were transferred into synchronised recipients after 48 h of in vitro culture in either SOFaaBSA (n=10) or on red deer epithelial oviduct monolayers (n=4). Five (33% 5/15) of the recipients that received 2- and 4-cell embryos were pregnant at Day 45 (verified by ultrasonography) and four recipients subsequently calved. One recipient receiving an embryo cultured in SOFaaBSA was pregnant at Day 45 and subsequently calved. The birth of five normal calves indicate that full developmental competence of red deer oocytes matured and fertilised in vitro can be achieved by the techniques described.     

Exposure of bovine oocytes to EGF during maturation allows them to develop to blastocysts in a chemically-defined medium

When cumulus-enclosed bovine oocytes were cultured for 24 h in serum-free medium containing 0 to 50 ng/ml EGF, the proportions of oocytes reaching metaphase II were higher (P < 0.05) in the presence of 30 ng/ml EGF (88.1 +/- 1.3%) than under control conditions (65.5 +/- 3.5%) or in the presence of 10 ng/ml (73.9 +/- 4.5%) and 50 ng/ml (73.6 +/- 4.0%) EGF. When oocytes matured under these conditions were inseminated in vitro, the proportions of oocytes penetrated were higher (P < 0.05) in 10 to 50 ng/ml EGF (96.7 +/- 3.3 to 100%) than in its absence (77.9 +/- 8.9%). However, the proportions of penetrated oocytes with male and female pronuclei did not differ among the different groups (96.7 +/- 3.3 to 100%). When oocytes were matured under the same conditions, fertilized in vitro, and cultured until 192 h post insemination in a chemically-defined medium, the proportion of embryos at the >/=2-cell stage was higher (P < 0.05) in the groups treated with 30 ng/ml (96.1 +/- 2.5%) and 50 ng/ml (90.6 +/- 3.5%) EGF than in the controls (71.8 +/- 3.1%) at 48 h post insemination. Although there were no differences in the proportions (37.3 +/- 5.3 to 47.2 +/- 5.8%) of >/=morulae at 144 h post insemination among treatments, the proportion of embryos developing to the blastocyst stage was higher (P < 0.05) in the presence of 10 to 50 ng/ml EGF (16.5 +/- 2.0 to 20.8 +/- 4.9%) than in control medium (3.4 +/- 2.1%). The mean blastocyst cell number at 192 h post insemination did not differ between culture media in the presence (91 to 107 cells) and the absence (116 cells) of EGF (10 to 50 ng/ml) during maturation. Thus, higher proportions of oocytes matured in serum-free medium with EGF than without EGF could develop to the blastocyst stage in a chemically-defined medium after in vitro fertilization. These results indicate that EGF can induce not only nuclear maturation but also cytoplasmic maturation of cumulus-enclosed bovine oocytes in vitro.     

Interrelationships of hormonal and ovarian responses in superovulated heifers pretreated with FSH-P at the beginning of the estrous cycle

The objective of this study was to determine the relationships between follicle stimulating hormone, (FSH), estradiol (E(2)), and progesterone (P(4)) concentrations in peripheral blood samples and the follicular dynamics prior to and during superovulation in heifers pretreated with FSH-P (10 mg, i.m.) (FSH-P-primed; n=9) or not (saline-primed; n=9) on Day 3 (Day 0 = estrus) of the estrous cycle. On Day 10, all heifers were superovulated with FSH-P (27.7 mg i.m.) in declining dosages over 5 days. Prior to and during superovulation, blood samples were collected one to five times daily, and the follicular dynamics were monitored daily by ultrasonography. Prior to superovulation, profiles of P(4) and E(2) did not differ (P>1) between the saline- and FSH-P-primed heifers. The FSH concentrations in saline-primed heifers decreased from 0.43 +/- 0.05 ng/ml to 0.30 +/- 0.04 ng/ml between Days 3 and 7 and then increased progressively to 0.59 +/- 0.04 ng/ml on Day 10. In contrast (P<0.002), FSH concentrations in the FSH-P-primed heifers remained constant between Days 3 and 10 and averaged 0.41 +/- 0.03 ng/ml. Higher increases in E(2) during superovulation (maximum values, 100 vs 46 pg/ml) and in P(4) after superovulation (maximum values, 39 vs 22 ng/ml) in the saline-than in the FSH-P-primed heifers reflected the greater increase in the number of follicles (>10 mm) and in the number of corpora lutea (CL) in the saline-primed heifers. Prior to the preovulatory luteinizing hormone (LH) peak during superovulation, there was a parallel (P>0.1) decrease in FSH concentrations in the saline- and FSH-P-primed groups. Within heifers partial correlations indicated that E(2) was correlated positively with the number of follicles (>/= 7 mm) and the size of the largest follicle during superovulation (r=0.54 to 0.81; P<0.01). Negative correlations were detected (P<0.01) between FSH and the number of follicles >/=7 mm prior to (r=-0.26) and during superovulation (r=-0.37). The results cofirm earlier reports indicating that priming with FSH-P decreases the superovulatory response in cattle. Interrelationships of hormonal and ovarian responses support the concept that the presence of large dominant follicles prior to superovulation limits the superovulatory response.     

Temporal aspects of ovarian follicular growth and steroidogenesis following exogenous follicle-stimulating hormone in Angus heifers

Ultrasonography and endocrine assay techniques were used to monitor structural and hormonal alterations made by the ovary in response to the biological actions of pituitary-derived follicle-stimulating hormone (FSH-P). Angus heifers (n = 36) were allotted to receive injections (twice per day) of either FSH-P (up to a total of 28 mg over a maximum of 4 days beginning on Day 10 of a synchronized estrous cycle) or saline in order to quantify temporal relationships among follicle growth and steroid hormone profiles. Transrectal ultrasonography was utilized at 12-h intervals to monitor and record follicle growth. Plasma was collected every 12 h for the first 48 h of the experiment and then every 6 h for the remainder of the experiment. At 48 and 60 h after the onset of treatments, prostaglandin F2α (PGF2α; 25 mg) was administered (i.m.). FSH-treated heifers (n = 6 at each time) were terminated at 24, 48, 72 and 96 h following the onset of treatment. Saline-treated heifers were terminated at 24 and 96 h (n = 6 at each time). After ovaries were obtained, follicular number and size were recorded and follicular fluid (FF) was collected. Plasma concentration of progesterone (P) and estradiol (E2) and FF concentration of P, E2, estrone, testosterone and androstenedione were determined by radioimmunoassays. Plasma concentration of E2 increased (P < 0.05) within 36 h of initiation of FSH treatment. Plasma P decreased (P < 0.0001) by 12 h post-PGF2α. Ultrasonographic examination revealed a significant decrease in the number of small follicles by 48 h, whereas the number of medium follicles increased (P < 0.05) by 60 h after the initiation of FSH treatment. The number of large follicles (LF ≥ 10 mm diameter) increased (P < 0.01) over the course of the experiment. The total number of ovarian follicles (TF) 24 h after the start of FSH treatment was correlated (r = 0.99; P < 0.0001) with the number of small follicles (SF ≤ 5 mm). At 72 h after the onset of FSH treatment, the number of medium follicles (i.e. 6–9 mm) was correlated with TF (r = 0.97; P < 0.0001). Estradiol was the predominant FF steroid. Follicular fluid E2 was greatest in follicles at 72 h after FSH treatment. Follicular fluid E2 and plasma E2 were positively correlated (r = 0.66; P < 0.001). Follicular aromatase activity was estimated by evaluating the ratio of FF estrogens (E) to androgens (A). Elevated aromatase activity (E:A ratio > 1.0) was detected in 196 of 206 follicles. The estrogen to progesterone ratio was used as an estimate of follicle viability. Eighty-five percent of the follicles were estimated to be viable (E:P ratio > 1.0). The peak E:A ratio in LF preceded by 24 h the peak concentration in FF E2 and plasma E2. In MF and SF the E:A ratio increased by 72 h. Enhancement of ovarian follicular growth (i.e. increased number and size of follicles; increased steroidogenesis) by exogenous, pituitary-derived FSH is characterized by (1) increased activity of aromatase, and (2) accumulation of FF E2, events which temporally preceded the increase in plasma concentration of E2. These observations will aid efforts to incorporate recombinant bovine FSH and somatotropin in an effort to develop more predictable superstimulation and ovulation induction protocols.