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.     

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.     

Effects of superovulation, culture and microinjection on development of rabbit embryos in vitro

Factors influencing the developmental potential of cultured rabbit zygotes and their ability to incorporate and integrate the WAP-hPC (human protein C) gene were investigated. Rabbit zygotes (n = 1053) were recovered from both superovulated and nontreated New Zealand White females. The hormonal treatment of rabbit donors resulted in a doubling of the number of recovered ova per donor when compared with the nontreated group (18 vs 9 ova). However, the quality of recovered zygotes (presence of both pronuclei) was significantly better in the nontreated group (99 vs 88%, Experiment 1). The effect of various culture media on the development of rabbit zygotes in vitro was evaluated after incubation under CO2-free conditions (Experiment 2). In serum-free, growth factor-supplemented medium (BSEITS, DME/F12, 1.5% BSA, EGF, insulin, transferrin and sodium selenite) the percentage of morula/blastocyst stage embryos was significantly higher (88%) than in DME/FCS, (DME/F12, 10% fetal calf serum, 59%) or the control group (DME/F12, 1.5% BSA, 25%). In Experiment 3, zygotes were microinjected with the WAP-hPC gene and were examined after 72 h of culture. Zygote cleavage and the percentage of morula/blastocyst stage intact embryos were higher (79 and 58%, respectively) than in microinjected embryos (31.0 and 21.5%, respectively). Summarized data of the PCR assay of microinjected zygotes demonstrated positive signals for the integration of the WAP-hPC gene in 6.6% (34 of 515) of all the microinjected zygotes.     

Superovulation of immature rats by continuous infusion of follicle-stimulating hormone

Immature female rats were infused s.c. continuously over a 60-h period with partially purified porcine pituitary follicle-stimulating hormone (FSH) preparations differing in degree of purity and having widely divergent luteinizing hormone (LH):FSH potency ratios as defined by radioreceptor assays. Rats infused with the more purified FSH preparation (FSH-A) ovulated a mean of 60-85 oocytes per rat on the morning of the third day (Day 1) after FSH infusion was begun (on Day -2). The same total dose of FSH administered as a single s.c. injection or as twice daily injections over the same 60-h period resulted in ovulation in only a minority of treated rats (3/16), with none achieving ovulation rates approaching those of rats infused continuously. High fertilization rates (80% of ovulated oocytes) were observed in superovulated rats joined with fertile males on the evening of the second day of infusion (Day 0). Of the 67 +/- 7 fertilized ova per rat retrieved from oviducts flushed on Day 1, 52 +/- 8, or 80%, were accounted for as morulae or blastocysts recovered when oviducts and uteri were flushed on the morning of Day 5, demonstrating essentially normal developmental rates and high survival rates in reproductive tracts of superovulated females during the preimplantation period. Infusion of rats with the same dose of a less well-purified FSH preparation (FSH-E) containing 20 times as much LH activity, or injection of rats with a superovulatory dose of pregnant mare's serum gonadotropin (PMSG) (40 IU), were much less effective in causing superovulation, with ovulation rates of 17 +/- 6 and 34 +/- 8 oocytes/rat, respectively, compared to 79 +/- 9 oocytes/rat infused with the FSH preparation (FSH-A) containing lower LH activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Embryo production from superovulated sheep inseminated with sex-sorted ram spermatozoa

An experiment was undertaken to assess the fertilizing capacity of sex-sorted, frozen-thawed ram spermatozoa, artificially inseminated into superovulated ewes, and the quality and survivability of the resultant pre-sexed embryos. Synchronized (intravaginal progestagen pessary and GnRH) donors were superovulated using PMSG and repeat ovarian stimulation with FSH before insemination. Ewes (n=67) were inseminated with either 30x10(6) or 15x10(6) motile non-sorted (control) or 15x10(6) motile sex-sorted (sorted) frozen-thawed spermatozoa (control: C30 or C15; sorted: S15, respectively) and the resultant embryos transferred immediately into synchronized recipients (n=160). The percentage of transferable embryos, pregnancy rate and embryo survival were similar (P>0.05) across all treatments. Oocyte cleavage rate was higher for ewes inseminated with S15 (172/230; 74.8%; P<0.05) than for C15 (97/151; 64.2%) or C30 (89/141; 63.1%) spermatozoa. Of the lambs resulting from embryos produced with sex-sorted spermatozoa, 86/93 (92.5%) were born of the predicted sex. This study demonstrated for the first time that pre-sexed offspring derived from superovulated sheep can be produced following transfer of embryos. Furthermore, sex-sorting by flow cytometry did not compromise the in vivo fertilizing capacity of ram spermatozoa in superovulated sheep, nor did it affect the quality or survivability of the resultant embryos.     

Rate of transport and development of preimplantation embryo in the superovulated buffalo (Bubalus bubalis)

This study was designed to ascertain the rate of transport and development of preimplantation embryo in the superovulated buffalo in order to determine the optimum time for their nonsurgical collection. Eighteen Murrah-type buffalo were superovulated with 600 mg NIH-FSH-P1. Luteolysis was induced by administration of PGF2 alpha at 72 (PG + 72) and 84 h (PG + 84) after initiating gonadotrophin treatment and fixed-time AI was done beginning at 36 h post PG + 72 administration and at 12-h intervals thereafter, upto 72 h. Six control buffalo received treatment similar to experimental group except that in place of FSH they received normal saline. For embryo collection, experimental animals were humanely killed at 6-h intervals corresponding to 156 (n = 2), 162 (n = 2), 168 (n = 2), 174 (n = 3), 180 (n = 3), 186 (n = 3) and 192 h(n = 3) after PG + 72 treatment, whereas the control animals were humanely killed at 156 (n = 2), 174 (n = 2) and 192 h (n = 2). Superovulated buffalo had higher number of ovulations than untreated controls (8.78 +/- 5.00 vs 0.67 +/- 0.51) and total ova/embryos recovered was 4.11 +/- 2.46 and 0.67 +/- 0.51, respectively. The high estradiol-17 beta (E2) levels with its prolonged rise may, by leading to reverse peristalsis in the oviduct with a consequent loss of some embryos in the peritoneal cavity, be one of the reasons for our inability to recover nearly 84/158 ova/embryos in the superovulated buffalo. In superovulated animals, nearly all the ova/embryos reached the uterus between 168 and 174 h post PG + 72 treatment or about 134 h (circa 5.5 d) after the onset of superovulatory estrus, suggesting that the ideal time for non-surgical embryo collection in the buffalo is between Days 7 to 8 after PG + 72 treatment or Days 5.5 to 6.0 of the superovulated cycle (estrus = Day 0). Embryo development of superovulated buffalo showed considerable variation as various stages of embryos (8 cell to expanded blastocyst) were recovered from the same donor buffalo, and the rate of development appeared to be 24 to 36 h faster than in cattle.     

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.     

Plasma hormone profliles and fertility in ewe lambs given progestagen supplementation after mating

Clun Forest ewe lambs (n = 124) were used to investigate the effects of post-mating progestagen supplementation on fertility. The animals were assigned to 1 of 3 three treatments: Group A (n = 41) served as the controls, Group B (n = 42) received 3 weekly injections of 6 mg of medroxyprogesterone acetate (MAP), while Group C (n = 41) was treated with intravaginal sponge containing 60 mg of MAP; all treatments were administered from Day 5 to Day 26 post mating. Supplementation did not increase the percentage of animals pregnant or those lambing: Group A, 72.2 and 66.6%; Group B, 57.5 and 50.0%; and Group C, 67.5 and 60.0%, respectively. Furthermore, there was no effect of supplementation on plasma progesterone, prolactin, cortisol, growth hormone, insulin, or glucose concentrations (P>0.05). However, pre- and post- mating hormone profiles differed significantly between the animals that lambed or aborted and the animals which were found to be barren at lambing. In the barren animals, progesterone concentrations were lower 4 days before and 9 to 33 days after mating (P<0.01), while overall prolactin concentrations were higher throughout the trial (P<0.01). But there was no difference between barren and fertile lambs in cortisol, growth hormone, insulin or glucose concentrations (P>0.05). These results indicate that progestagen supplementation does not increase the reproductive performance of ewe lambs. However, infertility is associated with reduced luteal function and increased prolactin concentration before and after mating.     

Early cycle FSH-p priming as a prelude to superovulating gonadotropin administration in ewes and heifers

The effect of an exogenous FSH treatment in the periovulatory, post-LH surge period on superovulatory response in the subsequent cycle of ewes and heifers was investigated. Thirty-five ewes were synchronized with progestagen pessaries and pregnant mares serum gonadotropin. The day following the onset of estrus (Day 1) 17 ewes received one intramuscular injection of 5 mg follicle stimulating hormone of porcine origin (FSH-p). All 35 ewes received another progestagen pessary on Day 1 and were superovulated with horse anterior pituitary extract (HAP). The ewes were bred and embryos collected 6 days following the onset of estrus. Early cycle FSH-p administration did not increase the subsequent ovulation rate (6.5 vs. 8.4 for controls, n.s.). Recovery rate for the FSH-p treated animals was higher (78.5% vs. 49.3%; P<0.05) as was fertilization rate (100% vs. 62.4%; P<0.05). The final result was a mean of 4.4 transferable embryos per ewe treated among the FSH-p boosted ewes and 2.6 transferable embryos per ewe treated among the control ewes.Twenty-nine heifers were brought into estrus with one 500-μg injection of prostaglandin F_2α (PG). Twelve of the 29 heifers were given one intramuscular injection of 10 mg FSH-p on either Day 2 or 3 (Day 1 is the day following the onset of estrus). All heifers were superovulated starting on Day 11–16, over a 4-day period using a decreasing dosage of FSH-p. Prostaglandin was administered at the time of the fifth superovulatory FSH-p injection and the heifers were bred by artificial insemination. Ova were recovered between 2 and 4.5 days following the onset of estrus. There was no effect on ovulation rate due to the interval from FSH-p priming to the day of superovulatory FSH-p initiation. The proportion of heifers that ovulated when given a FSH-p injection early in the cycle was higher than in the control group (94% vs. 68%; P<0.05). The primed heifers had a higher number of ovulations than did the control heifers (16.3 vs. 6.2; P<0.01). The effect of higher ovulation rate carried through all parameters measured, so that the FSH-p primed heifers also had a higher number of fertilized ova than the controls (10.7 vs. 3.9; P<0.05), indicating that there was no significant deterioration in ovum quality due to the FSH-p priming. The results show that FSH-p improved superovulatory efficiency in both sheep and cattle.     

Embryo transfer in cattle: Factors affecting superovulatory response,number of transferable embryos,and length of post-treatment estrous cycles

The effect of multiple ampules of frozen semen on conception rate in superovulated Holstein heifers was studied using 3 breeding regimens (n=25): 1 ampule at 12 hr (0 hr = beginning of estrus), 3 ampules at 12 hr, and 1 ampule at each of 0, 12, and 24 hrs. There was no significant effect of breeding regimen on recovered number of ova or percentage of fertilized ova. In another project, months during which heifers were superovulated with PMSG (5–8 heifers/month) did not significantly affect rate of superovulation (number of CL). Clinical records for 173 superovulatory treatments in 150 Holstein heifers were studied to obtain preliminary information on efficacy of treatment regimens, repeatability of response within heifers, and relationships between superovulation and length of estrous cycle; where indicated, contemporary, nontreated heifers were used for comparisons. Efficacy of PMSG vs FSH treatments did not differ in number of CL or number of ova recovered, but percentage of recovered ova that were transferable was greater (P<.05) for FSH (58.3) than for PMSG (42.9). There was an intraclass correlation coefficient of 0.33 (n=37) indicating repeatability within heifers in the magnitude of response to superovulatory treatment. Mean length (days) and coefficient of variation were significantly greater for superovulatory estrous cycles (cycles during which multiple CL were present; n, 141; mean, 31.2; SE, ± 1.2; CV, 46.2%) than for contemporary cycles in nontreated heifers (n, 63; mean, 20.8; SE, ± 0.4; CV, 13.9%). Treated heifers with short cycles (<15 days) had fewer CL (6.8 ± 1.4; mean ± S.E.) than heifers with intermediate cycles (15 to 27 days; 9.4 ± 0.6) or prolonged cycles (>27 days; 11.5 ± 0.7). Collection of an ovum from nontreated heifers resulted in shortening (P<.05) of the estrous cycle (n, 16; mean, 18.1 days) when compared to cycles from contemporary heifers in which collections were not done (n, 16; mean, 20.4 days).     

The efficiency of production, centrifugation, microinjection and transfer of one- and two-cell bovine ova in a gene transfer program

Forty crossbred beef heifers were superovulated with 2000 IU pregnant mare serum gonadotropin (PMSG) and mated twice by natural service during estrus. Ovulations were counted and ova were recovered during mid-ventral laparotomy between 44 and 54 h after the onset of estrus. The overall donor ovulation rate (M+/-SEM) was 15.2+/-1.3. There was a positive association between ovulation rate and the number of ova recovered (P<0.001), and between ovulation rate and the incidence of ova advanced beyond the two-cell stage of development (P<0.05). When grouped on the basis of superovulation response, the numbers (M+/-SEM) of recovered one-cell, two-cell and more advanced ova were 3.7+/-0.7, 1.0+/-0.3 and 0.5+/-0.3, respectively, for donors with up to 15 ovulations. The corresponding numbers for donors with more than 15 ovulations were 7.2+/-1.8, 6.0+/-1.3 and 2.8+/-1.2, respectively. Following centrifugation, pronuclei were visible in 68% of one-cell ova, and nuclei were visible in 80% of two-cell ova. Approximately 20% of ova were destroyed during DNA microinjection. A total of 66 centrifuged and DNA-injected ova were transferred to the oviducts of 26 crossbred beef heifers, each receiving two, three or four ova. Echography at Day 55 confirmed that 14 (54%) heifers were pregnant with 26 (39%) fetuses. Eleven heifers were held to calve and produced 21 calves.     

Superovulation of dairy cows with purified FSH supplemented with defined amounts of LH

This study investigated the effects of a purified follicle stimulating hormone (FSH) preparation supplemented with three different amounts of bovine luteinizing hormone (bLH) and a commercially available FSH with a high LH contamination on superovulatory response, plasma LH and milk progesterone levels in dairy cows. A total of 112 lactating Holstein-Friesian crossbred dairy cows were used for these experiments; the cows were randomly assigned to treatment groups consisting of purified porcine FSH (pFSH) supplemented with bLH. Group 1 was given 0.052 IU LH 40 mg armour units (AU) FSH (n = 6); Group 2 was given 0.069 IU LH (n = 32); Group 3 received 0.423 IU LH (n = 34); while Group 4 cows (n = 36) were superovulated with a commercially available FSH-P((R)). This compound appeared to contain 8.5 IU LH 40 mg AU FSH according to bioassay measurement. All animals received a total of 40 mg AU FSH at a constant dose twice daily over a 4-d period. Levels of milk progesterone and plasma LH were determined during the course of superovulatory treatment. The Group 1 treatment did not reveal multiple follicular growth, and no embryos were obtained. Superovulation of Group 3 cows resulted in significantly (P<0.05) more corpora lutea (CL; 12.6+/-1.1) and fertilized ova (5.1+/-1.3) compared with Groups 2 and 4 (10.1+/-0.9 and 2.6+/-0.6, 9.0+/-0.9 and 2.7+/-0.5, respectively). Due to a high percentage of degenerated embryos (33%) Group 3 yielded only one more transferable embryo than Groups 2 and 4. Among groups, LH levels differed in the period prior to induction of luteolysis and were similar thereafter. The progesterone pattern following FSH LH administration reflected the amount of LH supplementation. Milk progesterone levels on the day prior to embryo collection were correlated to the number of CLs and recovered embryos. It is concluded that under the conditions of our experiment superovulation with 0.423 IU LH 40 mg AU FSH may yield a significantly improved superovulatory response in dairy cows. It is further suggested that LH supplementation exerts its effects mainly on follicular and oocyte maturation during the period prior to luteolysis.     

Fertilization rates in superovulated and spontaneously ovulating mares

Embryo recovery per ovulation has been shown to be lower in superovulated mares than in untreated controls. The objectives of this study were to 1) determine whether follicles stimulated with superovulatory treatment ovulate or luteinize without ovulation, 2) determine fertilization rates of oocytes in oviducts of superovulated and control mares, and 3) evaluate viability of early stage embryos from superovulated and control mares when cultured in equine oviductal cell-conditioned medium. Cyclic mares were randomly assigned to 1 of 2 groups (n=14 per group) on the day of ovulation (Day 0): Group 1 received 40 mg of equine pituitary extract (EPE; i.m.) daily beginning on Day 5 after ovulation; mares assigned to Group 2 served as untreated controls. All mares were given 10 mg PGF(2alpha) on Day 5 and Day 6, and 3,300 IU of human chorionic gonadotropin (hCG) were administered intravenously once mares developed 2 follicles >/=35 mm in diameter (Group 1) or 1 follicle >/=35 mm in diameter (Group 2). Mares in estrus were inseminated daily with 1 x 10(9) progressively motile spermatozoa once a >/=35 mm follicle was obtained. Two days after the last ovulation the ovaries and oviducts were removed. Ovaries were examined for ovulatory tracts to confirm ovulation, while the oviducts were trimmed and flushed with Dulbeccos PBS + 10% FCS to recover fertilized oocytes. All fertilized oocytes (embryos) recovered were cultured in vitro for 5 d using TCM-199 conditioned with equine oviductal cells. Ninety-two percent of the CL's from EPE mares resulted from ovulations compared with 94% for mares in the control group (P>0.05). The percentages of ovulations resulting in embryos were 57.1 and 62.5% for EPE-treated and control mares, respectively (P>0.05). Eighty-eight (Group 1) and 91% (Group 2) of the freshly ovulated oocytes recovered were fertilized (P>0.05). After 5 d of culture, 46.4 and 40.0% of the embryos from EPE-treated and control mares developed to the morula or early blastocyst stage (P>0.05). In summary, the CL's formed in superovulated mares were from ovulations not luteinizations. Although embryo recovery was less than expected, fertilization rates and embryo development were similar (P>0.05) between superovulated and control mares.     

The effect of bovine pestivirus infection on the superovulatory response of Friesian heifers

The pathogenesis of reproductive loss associated with bovine pestivirus infection during the preovulatory period was investigated using superovulated heifers. Twenty-five Friesian heifers were selected and randomly assigned to either a control group (n = 12) which did not become infected or to a treatment group (n = 13) which became infected following intranasal instillation of 2 ml of serum inoculum containing 5.5 log(10) TCID(50)/ml non-cytopathic virus, 9 d prior to artificial insemination (AI). Transrectal ultrasonography was used to monitor follicular development and ovulation during the superovulatory period. Animals were superovulated using a standard protocol of twice-daily injections of FSH-P and then were inseminated twice commencing 12 h after the onset of estrus. The intensity of expression of estrus was higher in the control heifers than in the pestivirus-infected heifers. Of 13 pestivirus-infected heifers, only 3 heifers displayed standing estrus compared with that in the control group, in which 10 of 12 heifers exhibited standing estrus. The mean number of ova/embryos recovered from the control group heifers was 5.75 +/-2.31, of which 4.00 +/- 0.72 were evaluated as transferable quality embryos. In comparison, heifers in the pestivirus-infected group yielded only a mean of 0.60 +/-0.34 ova/embryos, of which 0.23 +/- 0.22 were transferable quality embryos. Based on ultrasonographic examination, 24 h after the first AI 82% of the presumptive ovulatory follicles had ovulated in the control group compared with an ovulation rate of only 17% in the treated group. The results of this experiment demonstrated that bovine pestivirus infection during the preovulatory period could adversely affect ovulation, thus leading to a significant reduction in the number of palpable corpora lutea and in the number and quality of embryos recovered.     

Fertilization rates in superovulating cows after deposition of semen on the infundibulum, near the uterotubal junction or after insemination with high numbers of sperm

Three experiments were conducted with 105 superovulating Holstein dairy cows in attempts to improve the fertilization rate. Cows were superovulated with follicle-stimulating hormone (FSH) and time of estrus was regulated with prostaglandin F(2)alpha (PGF(2)alpha). Semen was deposited on each infundibulum through a laparoscope inserted through the flank (Experiment 1) or near the uterotubal junctions through flexible tubing passed through the cervix and uterine horns (Experiment 2). In the third experiment, high numbers of sperm in fresh semen were deposited in the uterus. Cows were necropsied and ova were recovered and examined about 3.5 d after the beginning of estrus. Deposition of 0.5 ml of frozen-thawed semen on each infundibulum (Experiment 1) reduced both ovum recovery and fertilization. In ten cows inseminated on the infundibulum, ova representing 43% of ovulation points were recovered and 9% of these recovered ova were fertilized. In ten control cows, ova representing 80% of ovulation points were recovered and 62% of them were fertilized. In a 2 x 2 experiment with 36 superovulating cows (Experiment 2), 1 ml of diluted fresh or frozen semen was deposited either near the uterotubal junction or in the uterine body. The overall fertilization rate was 61%, with no significant effect of site of semen deposition or type of semen used. In Experiment 3, 2 or 3 ml of neat semen (average of 4.4 billion sperm) was deposited in the uterus of 12 cows; 183 of 197 intact ova (93%) were fertilized. In 56 control cows inseminated with 0.5 to 1.5 ml of frozen diluted semen (average of 70 million sperm), 502 of 947 intact ova were fertilized (53%, P<0.001). Insemination with high numbers of fresh sperm overcame problems of sperm loss or sperm transport and improved the fertilization rate.     

Pituitary response of prepuberal lambs to oestradiol-17 beta.

In two experiments 48 prepuberal Merino ewe lambs were injected with oestradiol-17 beta (E2) or saline to study the effect of E2 on their plasma LH levels and on oestrus and ovulation. In the three groups which received 30 (experiment I), 50 and 30 (experiment II) microgram E2 respectively, 27 out of 28 lambs showed an LH response, the corresponding mean LH peaks being 64.3 +/0 22.5, 153.6 +/-33.4 and 91.7 +/- 16.9 ng/ml at mean intervals of 11.1, 11.2 and 10.5 h, respectively, after injection. None of the 20 lambs in the control groups had an LH level higher than 18 ng/ml 12 h after injection. In the three E2 groups, 41.7, 62.5 and 37.5% of animals showed oestrus within 26 h of injection while in the control groups only one animal showed oestrus. Of 13 animals showing oestrus in the E2 groups, 11 failed to ovulate. The mean pre-injection plasma FSH level in experiment I was 102.7 ng/ml, and in four 5--7-month-old lambs over several weeks uas 155.3 ng/ml. Despite these high pre-injection levels of FSH, it appears that the follicles were unable to respond to the LH peak which followed the E2 injection.     

Endocrine responses of goats after induction of superovulation with PMSG and FSH

Goats in Group A were pretreated for 9 days with a synthetic progestagen, administered via intravaginal sponge, and 1000 i.u. PMSG s.c. on Day 12 of the oestrous cycle. Goats in Group B had the same PMSG treatment, but not the progestagen pretreatment. Group C goats received a s.c. twice daily injection of a porcine FSH preparation (8 mg on Day 12, 4 mg Day 13, 2 mg Day 14 and 1 mg Day 15). Oestrus was synchronized in all animals by 50 micrograms cloprostenol, 2 days after the start of gonadotrophin treatment. The vaginal progestagen sponges were removed from Group A at the same time. Mean ovulation rate was slightly higher in FSH-treated than in the PMSG-treated animals, whereas the incidence of large follicles that failed to ovulate was significantly elevated in PMSG-treated animals in Group B. More goats in Groups A and B than in Group C exhibited premature luteal failure. Progestagen pretreatment appeared to suppress both follicular and luteal activity, as indicated by numbers of large non-ovulating follicles and by the magnitude and duration of elevated plasma oestradiol levels following PMSG stimulation, and by decreased plasma progesterone levels before and after PMSG treatment. Oestrogenic response to FSH was considerably less than that to PMSG, as indicated both by a considerably shorter duration of elevation of circulating oestradiol levels during the peri-ovulatory period, and by lower maximal oestradiol levels. Differences in the ovarian responses to PMSG and FSH may be attributed primarily to differences in the biological half-life of each preparation.     

Preimplantation embryo development and serum steroid levels in immature rats induced to ovulate or superovulate with pregnant mares' serum gonadotropin injection or follicle-stimulating hormone infusions

Follicular stimulation protocols using pregnant mares' serum gonadotropin (PMSG) or a follicle-stimulating hormone (FSH) preparation were compared to evaluate the yield and quality of embryos obtained from immature rats. Rats received a superovulatory dose of PMSG (401U), a nonsuperovulatory dose of the same gonadotrophin (4 IU), or a continu ous s.c. infusion over a 72-h period with a purified FSH preparation containing an opti mum ratio of luteinizing hormone (LH): FSH (FSH-hCG). The females were caged with fertile males on the evening of the 3rd day of gonadotropin treatment and scored for the occurrence of mating on the next morning; subgroups were killed on days 1–4 of preg-nancy. High fertilization rates were observed in rats treated with 4 IU PMSG (84.1%) and in rats infused with FSH-hCG (91.0%); however, a much lower fertilization rate was observed following treatment with 40 IU PMSG (41.5%). From median ovulation rates of 9 and 79 in rats treated with 4 IU PMSG and in rats infused with FSH-hCG, medians of 8 and 69 embryos, respectively, were recovered from reproductive tracts flushed on day 4 of pregnancy, from which 75% were morulae or blastocysts; in contrast, from a median ovu lation rate of 42.5, a median of only 12 embryos was recovered on day 3 of pregnancy following superovulation with 40 IU PMSG of which 80% were degenerate ova. Serum steroid profiles during the first 4 days of pregnancy differed significantly among treatment groups, the major differences being in substantially elevated levels of estradiol and andro-gens on days 1–3 in rats receiving the high (40 IU) dose of PMSG. Levels of these steroids in rats superovulated with the FSH-hCG infusion regimen were only marginally elevated above levels observed in rats treated with the low (4 IU) nonsuperovulatory dose of PMSG. Consistent with high ovulation rates, serum progesterone levels rose to considera bly higher levels during the period in both superovulated groups than in animals receiving the low, nonsuperovulatory dose of PMSG. This work describes a novel method to superovulate rate (FSH-hCG) leading to high yields of normally developing embryos at all preimplantation stages and illustrates the close association between high yield of emyryos and low levels of circulating andorgens and estradiol-17β during the preimplantation period.     

Ovulation of aged follicles does not affect embryo quality or fertility after a 14-day progestagen estrus synchronization protocol in ewes

The aim was to examine the effect of ovulation of aged follicles on embryo quality and fertility in ewes. In Experiment 1, ewes (n = 39) received a prostaglandin analogue on Day 6 of the cycle and then received either a progestagen sponge from Day 6 to 20 after estrus (Single sponge) or a progestagen sponge on Day 6 that was replaced on Day 11 and 16 and removed on Day 20 (Multiple sponges). In a subgroup of ewes, the growth of ovarian follicles was characterised using ultrasonography. Fertile rams were introduced 48 hours after sponge withdrawal; we slaughtered the ewes on Day 5 of pregnancy and recovered the embryos. The mean age of the ovulatory follicles was greater in ewes that received a single sponge compared with multiple sponges (8.7+/-0.8 days, range 4 to 14, versus 4.5+/-0.7 days, range 3 to 6; P<0.05). However, the groups did not differ (P>0.05) in ovulation rate (2.4+/-0.3 corporal lutea per ewe) or the proportion of good quality embryos recovered (71 to 82%; developed to the early morula stage or further). In Experiment 2, ewes (570 in total) received treatments similar to those in Experiment 1 but were kept until lambing. Ewes that received a single sponge came into heat earlier (P<0.05) than those that received multiple sponges, but > or = 97% of ewes in all groups (P>0.05) were bred by 48 to 72 hours after ram introduction. There was no difference (P>0.05) between groups for the proportion of ewes that lambed to first service (80 to 86%) or the number of lambs per ewe (1.94+/-0.08 lambs). We conclude that when luteolysis occurs at the beginning of progestagen synchronisation, ewes will ovulate aged follicles, but that compared to shorter duration follicles, these follicles produce oocytes that are equally competent to be fertilised and develop into good quality embryos and full-term lambs.     

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.