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.     

Cervical versus intrauterine insemination of ewes using fresh or frozen semen diluted with aloe vera gel

This study was conducted at Belen de Escobar, Argentina, in March and April 1987. Experimental work on synchronization of estrus, deep-freeze conservation of ram semen and small fertility trials involving cervical and intrauterine (i.u.) insemination methods was undertaken. A total of 80 Corriedale ewes were used in seven insemination trials. Insemination trials were grouped into two experimental groups for comparison of 1) frozen semen diluted with an experimental extender and a control diluent inseminated cervically or i.u. in synchronized/superovulated ewes and 2) cervical insemination of fresh diluted or frozen semen in ewes inseminated at natural estrus or in ewes that were synchronized/superovulated. An overall ovulation rate of 8.7 +/- 0.5 was obtained by using a superovulatory regimen consisting of 3 mg Norgestomet implants and a total dose of 18 mg follicle stimulating hormone-pituitary (FSH-P). Numbers of ova recovered per ewe following superovulation ranged from 4.3 to 5.4. In experimental Group I, fertilization rates improved when laparoscopic intrauterine AI was used compared with cervical insemination (P<0.05). Fertility rates of i.u. and cervical insemination of frozen semen diluted with the experimental extender showed satisfactory fertilizing capacity. In experimental Group II, a lower number of fertilized ova were recovered from ewes inseminated with frozen semen (P<0.02), irrespective of their estrus manipulation.     

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.     

Intrauterine insemination enhances fertility of frozen semen in superovulated ewes

Superovulated ewes were inseminated with fresh or frozen semen in a factorial experiment which compared two techniques of artificial insemination; i.e. conventional cervical deposition and intrauterine deposition at laparoscopy. Similar fertilization rates resulted from insemination with fresh semen at cervical (81% of ova from 11/11 ewes) and intrauterine (83% of ova from 10/12 ewes) sites. These results approached those observed in a naturally-mated group (95% of ova from 5/5 ewes). In ewes inseminated with frozen semen, fertilization rate was markedly reduced (P less than 0.05) after cervical insemination (11% of ova from 3/11 ewes) and partly restored (P less than 0.05) after intrauterine insemination (50% of ova from 8/11 ewes).     

Developmental ability of in vitro matured sheep oocytes collected during the nonbreeding season and fertilized in vitro with frozen ram semen

During the nonbreeding season, oocytes recovered from ovaries of FSH-primed or nonprimed ewes were matured in the presence or absence of granulosa cells collected from ovaries of primed or nonprimed ewes prior to in vitro fertilization with either fresh or frozen-thawed sperm. Following fertilization, ova were cultured for 24 h in synthetic oviduct fluid medium (SOF) supplemented with 20% human serum at 39 degrees C under humidified 5% CO(2), 5% O(2), 90% N(2) and then assessed for cleavage. Overall, 52% of ova cleaved. Cleavage was not affected by the source of sperm. Significantly more oocytes from primed follicles cleaved after 24 hours than those from nonprimed follicles (P<0.001). Maturation of oocytes in the presence of granulosa cells from nonprimed ewes resulted in a lower cleavage rate (44%, P<0.05) than in the presence of granulosa from primed ewes (59%) or no granulosa cells (50%). Oocytes (n = 508) from primed ewes were matured in the presence of granulosa cells (also from primed ewes) and fertilized in vitro with frozen-thawed sperm. Following in vitro culture for 24 hours, 68 of the 270 (53%) cleaved embryos were transferred to 17 recipient ewes, 15 of which remained pregnant to term, producing 24 lambs. The remaining 202 cleaved embryos were cultured for a further 5 days, of which 73 appeared to reach the morula/blastocyst stage and 61 were transferred to 16 recipients. Two ewes remained pregnant to term producing two lambs. These results demonstrate that production of sheep embryos using in vitro maturation and fertilization techniques is possible in the nonbreeding season. However, the poor viability of embryos obtained following extended culture needs to be resolved before such techniques can be usefully applied.     

Superovulation of a low fecundity sheep breed using a porcine gonadotrophin extract with a defined LH content (Pluset)

The aim of this study was to determine the efficiency of a porcine pituitary gonadotrophin extract with a defined pLH content in the superovulation of sheep. Estrus was synchronized in 61 Polish Mountain ewes with intravaginal fluorogestone acetate sponges. Twenty-four hours before the sponges were removed, the ewes underwent different superovulatory treatments: Group I 250 IU of pFSH with 250 IU of pLH (n=19); Group II 500 IU of pFSH with 500 IU of pLH (n=19); and Group III 750 IU of pFSH and 750 IU of pLH (n=18). Gonadotrophine was administered intramuscularly twice a day over a 3-day period in decreasing dosages. A control group of ewes (n=5) was treated with saline. In most of the ewes estrus began about 20 hours after sponges were removed. All the ewes were bred naturally every 12 hours. Superovulation was confirmed in 75% of the treated animals. The ewes receiving 250 IU each of pFSH and pLH produced an average of 7.6 +/- 3.1 corpora lutea (CL), 6.3 +/- 2.4 ova and 4.3 +/- 4.1 transferable embryos. Group II (500 IU of pFSH and pLH) produced 8.5 +/- 4.0 CL, 7.6 +/- 4.1 ova, and 4.1 +/- 2.9 transferable embryos. Group III (750 IU each of pFSH and pLH) produced 8.3 +/- 5.2 CL, 7.5 +/- 5.5 ova and 5.2 +/- 5.1 transferable embryos. The mean embryo recovery rate was 87% for all three groups. Differences in superovulatory response and embryo recovery rate among the groups were not statistically significant (P>0.05).     

The extent and timing of prenatal loss in gilts

The potential litter size of gilts that is based on the ovulation rate is much higher than the actual litter size, which depends on the fertilization rate and subsequent prenatal mortality. Prenatal mortality is divided into embryonic mortality (before Day 30) and fetal mortality (after Day 30). Prenatal loss includes both fertilization failure and prenatal mortality. Crossbred gilts (n = 149) were bred at the first observed estrus after being exposed to the boar at 200 days of age. Time of the first insemination after estrus detection was determined by measurement of vaginal conductivity using a Walsmeta meter. A second insemination was administered either 8 or 16 hours later. Artificial insemination with fresh semen (0 to 3 days old) was used throughout the experiment. Gilts were slaughtered on Day 3 (n = 26), Day 10 (n = 42), Day 30 of gestation (n = 45) or they were allowed to farrow (n = 36). Gilts slaughtered on Day 3 were used to estimate the fertilization rate. Gilts slaughtered on Day 10 and Day 30 were used to calculate embryonic mortality, while fetal mortality was calculated from the gilts that farrowed. The mean (+/-SEM) number of corpora lutea (CL) was 13.15+/-0.46, 13.36+/-0.37 and 12.97+/-0.39 for gilts slaughtered at Days 3, 10 and 30, respectively (P>0.05), and the mean (+/-SEM) number of normal embryos recovered was 11.12+/-0.69, 9.46+/-0.55 and 9.33+/-0.58, respectively. Litter size at parturition was 9.10+/-0.54. There was a significant difference between the number of normal embryos on Day 3 and Day 30 (P=0.05) and also between the number of normal embryos at Day 3 and the number of piglets at term. Ninety percent of the ova were recovered at Day 3. The fertilization rate was calculated either 1) assuming that unrecovered ova had a similar fertilization rate as the recovered ova (FRER=94.5+/-2.0%) or 2) assuming that unrecovered ova were unfertilized (FROR=84.5+/-2.5%). It was concluded that FRER was a more accurate estimation of the fertilization rate. Based on this fertilization rate, embryonic mortality between Day 3 and Day 10 was 20.8+/-8.3%, with an additional 12.5+/-7.1% loss between Day 10 and Day 30, when all gilts were included (P = 0.308). Thus the total prenatal loss, including fertilization failure, up to Day 10 was 26.3% and to Day 30 it was 38.8%. Fetal mortality was 2.2%, giving a total prenatal mortality (excluding fertilization failure) of 35.5% and a prenatal loss of 41%. Most of the prenatal loss was due to embryonic mortality. In those gilts that remained pregnant most of the embryonic loss occurred before Day 10 (19.0+/-6.3%; P=0.003). There was no further loss between Day 10 and 30 of pregnancy. There was a significant difference between the loss from Day 3 to Day 10 compared with the loss from Day 10 to Day 30 (P=0.05); therefore, most of the embryonic loss in pregnant gilts occurred before Day 10. Since fetal mortality was 3.2+/-6.3%, most of the prenatal loss was due to embryonic mortality.     

Laparoscopy for intrauterine insemination and embryo recovery in superovulated ewes at a commercial embryo transfer unit

Of 111 variable age, pedigree ewes subjected to a range of superovulatory regimens and then submitted to embryo recovery by laparoscopy, nine had adhesions corresponding to a mid-line laparotomy (presumably from a previous attempt to recover embryos) and could not have their embryos recovered by the laparoscopic technique. Of the remainder, 27 ewes (26.5%) had less than three ovulations or had prematurely regressing corpora lutea at the selected time for embryo recovery (Days 5 to 6 following insemination), and no attempt was made to recover embryos from them. For the 75 ewes subjected to laparoscopic ovum recovery following laparoscopic intrauterine insemination, the average number of ovulations (+/- SEM) was 7.9 +/- 0.6; the average ovum recovery (mean of values for each ewe) was 51.7% +/- 3.5; and the percentage of recovered ova that were fertilized was 87.3%. For a further nine 3-yr-old crossbred ewes the mean values for ovulation and ovum recovery were 7.6 +/- 1.2 and 70.1 +/- 7.7, and were not significantly different for the two insemination methods used (laparoscopic intrauterine vs cervical). In general, ovulation rates for ewes given pregnant mare serum gonadotrophin (PMSG) tended to be lower (5.2 +/- 0.7) than for those given porcine follicle stimulating hormone (pFSH, 7.7 +/- 0.8) or human menopausal gonadotrophin (hMG, 7.7 +/- 2.3). Ova recovery rates were similar on Days 5 and 6 (Day 0 = insemination), and were not affected by method of insemination (laparoscopic intrauterine vs cervical).     

The use of synthetic gonadotropin releasing hormone treatment in the collection of sheep embryos

Gonadotropin releasing hormone (GnRH) treatment was examined as a means of improving the efficacy of embryo collection in the sheep following intrauterine insemination of frozen-thawed semen. In summary, treatment consistently improved fertilization rates and the number of fertilized ova collected per ewe was enhanced compared with untreated ewes. The yield of fertilized ova in ewes treated with follicle stimulating hormone (FSH) was maximized by administering GnRH 36 h after progestagen treatment; 24 h was the preferred time in ewes treated with pregnant mare serum gonadotropin (PMSG). There was a significant (P < 0.001) increase in the percentage of unfertilized ova in the former treatment when GnRH was given at 24 h. An examination of the time of insemination (0, 6, 12 and 18 h before the median time of ovulation) indicated that fertilization rates were highest when insemination occurred at 6 h in both GnRH-treated ewes and in untreated ewes. In GnRH-treated ewes, the recovery of ova was lowest when insemination occurred at the time of ovulation. The number of motile frozen-thawed spermatozoa required for fertilization following treatment was estimated to be approximately 20 x 10(6) per uterine horn. GnRH-treatment also improved the yield of fertilized ova in sheep that were naturally mated, although this yield was lower than that obtained with intrauterine insemination of frozen-thawed semen. It is concluded that fertilization failure, a major problem in sheep embryo collection, can be eliminated through judicious use of GnRH treatment and properly timed intrauterine insemination.     

Development of bovine embryos in a cell-free culture medium: Effects of type of serum, timing of its inclusion and heat inactivation

Bovine embryos, derived from in vitro matured (IVM)/in vitro fertilized (IVF) ova, were used to investigate the effects of timing of serum inclusion in the culture medium and different types of blood sera and heat inactivation of the serum on embryo development. In Experiment 1, oocytes at 18 h post insemination were allocated to 1 of the following 4 treatments: 1) TCM-199 + 0.1 mg/ml polyvinylalcohol (PVA), 2) TCM-199 supplemented with 10% bovine calf serum (BCS), 3) PVA medium followed by BCS medium at 47 h, or 4) PVA medium followed by BCS medium at 82 h. Supplementation with BCS at 18 h post insemination suppressed (P<0.05) development of morulae/blastocysts (17.6%) when compared with PVA (30.5%) or with serum supplementation at 47 or 82 h post insemination (32.4 and 27.6%, respectively). However, inclusion of BCS at 18, 47 or 82 h post insemination produced more blastocysts (16.8, 29.3 and 22.1%, respectively; P<0.05) than medium +PVA (8.8%). In Experiment 2, ova were cultured from 18 h to 42 h post insemination in PVA-medium, then >/=2-cell embryos were transferred into serum-supplemented medium for another 168 h. Fetal bovine serum (FBS) +/- heat-inactivation (56 degrees C for 30 min, = heated FBS) suppressed morula/blastocyst development compared with medium + PVA, medium + BCS or medium + heated BCS (P<0.05). Bovine calf serum was superior to FBS in supporting blastocyst development (35.1 and 15.2%, respectively), but there was no difference between BCS and heated BCS. However, heated FBS increased the proportion of blastocysts/>/=8-cell embryos compared with that of FBS (51.0 and 31.4%, respectively; P<0.05). These results indicate that the type of serum supplementation and the timing of its inclusion in the culture medium markedly affect bovine embryo development in vitro, and that heat inactivation of serum with high embryotrophic properties is not necessary.     

The difference in embryo quality between Belclare and Suffolk ewes is not due to differences in oocyte quality

We have previously reported that the percentage of fertilized oocytes which reached the blastocyst stage by Day 6 after AI with frozen-thawed semen was higher for Belclare (94%) than Suffolk (59%) ewes. This may reflect differences in the timing of fertilization (Experiment 1) or differences in oocyte quality (Experiments 2 and 3). In Experiment 1, oocytes recovered from slaughterhouse ovaries were matured in vitro for 18, 20, 24, 28 or 30 h prior to fertilization and were then cultured in vitro. In Experiment 2, Belclare (n = 69) and Suffolk (n = 71) ewes were laparoscopically inseminated using frozen-thawed semen. Presumptive zygotes were recovered between 23 and 47 h post-insemination and cultured in vitro (grouped by breed). In Experiment 3, immature oocytes from Suffolk and Belclare ewes, were matured, fertilized and cultured in vitro (grouped by breed). Cleavage rate and blastocyst development was assessed. There was no effect of time of fertilization on cleavage rate, however, a lower proportion of cleaved oocytes reached the blastocyst stage after insemination at 30h compared to 24 h (P < 0.001). Ewe breed did not affect cleavage rate of oocytes matured and fertilized in vivo (41+/-9.6 and 47+/-10.1) or in vitro (47+/-9.4 and 52+/-9.4) for Belclare and Suffolk ewes, respectively (P > 0.05; %+/-S.E.). Likewise, ewe breed had no effect on the percentage (+/-S.E.) of cleaved oocytes developing to the blastocyst stage for in vivo (29+/-7.2 and 25+/-7.9) or in vitro matured and fertilized oocytes (29+/-6.1 and 36+/-5.9) from Belclare and Suffolk ewes, respectively (P>0.05). Based on this study oocyte quality does not differ between the breeds and in addition a 4h difference in the timing of fertilization, reflective of the breed difference in the timing of the LH surge in vivo, would not affect early embryo development.     

Season affects characteristics of the pre-ovulatory LH surge and embryo viability in superovulated ewes

The aim of this study was to determine whether there are seasonal shifts in ovulatory response, and in the viability of ova recovered from superovulated ewes. Fifty mature ewes underwent a standard oestrous synchronisation (CIDR), superovulation (oFSH) and artificial insemination procedure during October (peak breeding season) and April (transition to anoestrus). In each month peripheral LH and progesterone concentrations were measured around the time of ovulation and embryos were recovered, graded and cryopreserved on day 6 after insemination. During the subsequent breeding season, grade 1 and 2 morulae and unexpanded blastocysts were thawed and transferred singly to synchronous recipients (October, n = 40; April, n = 40) or cultured in vitro for 18-20 h (October, n = 107; April, n = 98). Following culture, viable embryos were stained to count cell nuclei or assayed to measure their capacity for glucose metabolism ([3H]glucose) and protein synthesis ([35S]methionine). Peak LH concentrations were higher in October than in April (38.2 +/- 3.26 ng ml(-1) versus 25.7 +/- 1.99 ng ml(-1), respectively; P < 0.01) and the pre-ovulatory LH surge was advanced by approximately 3 h (P < 0.05). Progesterone concentrations at CIDR withdrawal were lower in October than in April (3.1 +/- 0.16 ng ml(-1) versus 4.3 +/- 0.19 ng ml(-1), respectively; P < 0.001) but were not different at embryo recovery. Season did not affect the numbers of corpora lutea per ewe or the numbers of ova recovered but the proportion of recovered ova that was unfertilised/degenerate was lower in October than in April (0.43 versus 0.58, respectively; P < 0.001). For embryos containing more than 16 cells, there was no effect of season on the median stage of development or morphological grade. The proportions of October and April embryos that established pregnancy following transfer to recipient ewes were 0.78 and 0.70 (not significantly different), and that were viable after in vitro culture were 0.66 and 0.37 (P < 0.05), respectively. Season did not affect the number of nuclei per viable embryo or the capacity for protein synthesis but the glucose uptake of October embryos was approximately double that of April embryos (3163+/-293.4 dpm versus 1550+/-358.9 dpm, respectively; P < 0.05). Results indicate that during the late compared to peak breeding season, there is an increased incidence of fertilisation failure as a possible consequence of seasonal shifts in LH secretion and (or) associated effects on follicular function. Frozen-thawed embryos produced at contrasting stages of the breeding season are equally viable in vivo but those produced during the late, as opposed to the peak breeding season have lower viability following in vitro culture.     

Effect of freezing rate of ram spermatozoa on subsequent fertility in vivo and in vitro

Ram spermatozoa are most susceptible to damage during freezing between the temperatures of -10 degrees C and -25 degrees C. The objectives of the present study were to examine how freezing rate through this critical temperature zone affected the fertility of spermatozoa as assessed in vivo and in vitro. Semen from six adult rams was frozen at two different rates ("fast": 5 degrees C/min from +5 to -25 degrees C; "slow": 0.5 degrees C/min from +5 to -25 degrees C). In Experiment 1, semen from the fast and slow treatments was used to fertilize ovine oocytes that had been matured in vitro. Semen from the fast treatment yielded a higher cleavage rate (57% vs. 26%; P<0.001) and more blastocysts per oocyte (28% vs. 13%, P<0. 001) than slow-frozen. No correlation was found between fertilizing ability and viability as assessed by fluorescent probes. Experiment 2 was designed to establish the conception rates following both cervical and intrauterine insemination of frozen-thawed semen from the same bank of semen as used in Experiment 1. Ewes were superovulated with FSH and inseminated by laparoscopy with frozen semen. A significant difference was found in the number of fertilized ova following embryo recovery (81.4% vs. 39.3%; P<0.001). In a further study, 119 mature cull ewes were inseminated following a 12-day synchronization treatment with frozen semen by either intrauterine (laparoscopic) or cervical insemination. Insemination with fast-frozen semen resulted in a significantly higher pregnancy rate (P<0.05) irrespective of method of insemination. The data show that freezing rate affects the proportion of spermatozoa that retain their fertilizing ability post-thawing. However, once fertilization has occurred, development to the blastocyst stage is independent of freezing rate.     

Effects of melatonin and undernutrition on the viability of ovine embryos during anestrus and the breeding season

This study examined the effects of melatonin and level of nutrition on embryo yield during anestrous and breeding season. Adult Rasa Aragonesa ewes were assigned randomly to one of the four treatment groups in two experiments using a 2x2x2 factorial design. Individuals were treated (+MEL) or not treated (-MEL) with a subcutaneous implant of melatonin for 42d (Melovine, CEVA) and fed 1.5 (control, C) or 0.5 (low, L) times the daily maintenance requirements for 20d. Ewes were mated at oestrus (Day=0) and embryos were recovered on Day 5. Level of nutrition and melatonin supplements did not have a significant effect on ovulation rate or the number of recovered ova per ewe in the Reproductive Season (RS) and the Anestrous Season (AS). During the RS, undernutrition reduced the number of viable embryos per ewe (C: 1.1+/-0.2; L: 0.6+/-0.2; P<0.05); however, the number of viable embryos per ewe in the L+MEL group (0.2+/-0.15) was significantly lower than it was in the L, C+MEL and C groups (0.9+/-0.3, 1.2+/-0.3, 1.0+/-0.4, respectively; P<0.05). In the AS, nutrition did not have a significant effect on the number of viable embryos per ewe, although melatonin supplements might have improved rates slightly. Embryo viability rate (% viable embryos/embryos recovered) was unaffected by melatonin supplements or level of nutrition in the RS and the AS. Season had a strong effect on the number of viable embryos per functional corpus luteum among ewes in the L+MEL group, only (RS: 0.2+/-0.1; AS: 0.6+/-0.2; P<0.05). In conclusion, undernutrition impaired the viability of sheep embryos in the RS, particularly among ewes that were given melatonin supplements subcutaneously, but melatonin appeared to improve embryo quality in the AS, which suggests that the mechanisms involved in the interactive effects of melatonin and nutrition on embryo development are influenced by season.     

Estrus synchronisation and fertility in gilts using a synthetic progestagen (Allyl Trenbolone) and inseminated with fresh stored or frozen semen

An orally active synthetic progestagen was administered at two dosage levels to synchronise estrus in gilts. Fertility following insemination with either fresh stored or frozen semen was determined by examining surgically recovered ova for cleavage, and numbers of spermatozoa attached to the zona pellucida, or enumeration of embryos in gilts slaughtered 30 days post insemination. There was no significant difference (P>0.05) between treated and control groups in the duration of estrus or in fertility as determined by cleavage of ova. A significantly (P<0.001) shorter interval to estrus and better synchronisation was obtained with both treatment groups than with the control group. The mean interval from the end of treatment to the onset of estrus for the untreated controls and the treated groups receiving 12.5 and 15 mg compound per day was 11.25 +/- 10.4 SD; 5.6 +/- 0.52 SD and 7.3 +/- 5.3 SD. Fresh semen yielded significantly (P<0.01) more cleaved ova than frozen semen.     

Cross fertilization in vivo and in vitro between three species of vesper mice, Calomys (Rodentia, Cricetidae)

Cross fertilization was tested between oocytes of Calomys callidus and spermatozoa from C. callidus, C. musculinus and C. laucha by both in vivo and in vitro insemination. After in vivo and in vitro insemination, respectively, percentages of oocytes fertilized were 68.8 and 46.6 (C. callidus X C. callidus), 20.3 and 12.7 (C. callidus X C. musculinus), 26.7 and 4.3 (C. callidus X C. laucha). Thus, the percentages obtained after in vitro insemination were always lower than those obtained with in vivo insemination. It was found that 23.9% and 44.4% of two-cell hybrid embryos were present in oviducts 30 hr after in vivo insemination of C. callidus females with C. musculinus or C. laucha spermatozoa, respectively. At a later stage (56 hr postinsemination), development did not progress further, and abnormal embryos were found both at 30 and 56 hr postinsemination, suggesting some kind of cleavage arrest or degeneration of the embryos. We suggest that fertilization is not strictly species-specific, at least among the species we studied, but that there are some factors that reduce the efficiency of interspecific fertilization.     

Effects of once- versus twice-weekly transvaginal follicular aspiration on bovine oocyte recovery and embryo development

Ultrasound-guided transvaginal follicular aspiration combined with in vitro maturation/in vitro fertilization (IVM/IVF) and culture was used to obtain bovine preimplantation stage embryos. Evaluated were the effects of aspiration frequency on oocyte recovery and embryo development following IVM/IVF. In Experiment 1, transvaginal follicular aspiration was performed once (n=5) or twice (n=5) weekly in multiparous Angus cows with the aid of a transvaginal sector transducer (5-MHz). In Experiment 2, aspiration was performed on Angus cows once weekly (n=6), twice weekly (n=4), or twice weekly after treatment with FSH (15 mg; n=4). Follicles (>2 mm) were punctured using a 55-cm needle (17g), and oocytes were aspirated through the needle and silastic tubing (2 m) by vacuum suction (75 mmHg). The oocytes were examined for morphology and were in vitro matured and fertilized. Following IVF, all ova were co-cultured in vitro for 7 d on Buffalo Rat liver cells. Oocyte recovery rates per asp?ration session in Experiment 1 were not different between groups aspirated once or twice weekly (6.8+/-2.0 vs 6.3+/-1.1 oocytes/session; x+/-SEM) or in Experiment 2 between groups aspirated once, twice, or twice plus FSH treatment (7.7+/-1.8 vs 9.5+/-1.1 vs 6.2+/-1.1; P>0.10). In vitro development to the blastocyst stage was not different between the once, twice or twice-weekly aspiration plus FSH treatments or control oocytes obtained from cows at slaughter (23.1 vs 26.1 vs 18.0 vs 27.9%; P>0.10). Oocytes from the twice-weekly and twice-weekly plus FSH aspiration groups generated a higher percentage of Grade-1 quality embryos than the once-weekly group (P<0.05). In commercial bovine oocyte aspiration, more transferable embryos can be generated from twice-weekly aspirations than from once-weekly aspiration.     

Progesterone and 17beta-estradiol, but not follicle stimulating hormone, alter the sex ratio of murine embryos fertilized in vitro

The objective was to determine the effects of adding progesterone, 17beta-estradiol (17beta-E2), and FSH during in vitro fertilization on development and sex ratio of murine embryos. Progesterone (33-330 pg/mL), 17beta-estradiol (17beta-E2); 10-70 pg/mL), and FSH (0.01-0.05 IU/mL), were added to human tubal fluid (HTF); this medium (with or without hormones) was used to pre-incubate sperm (2h) and to co-incubate sperm and oocytes (6h). Thereafter, the ova were washed and incubated in mM16 medium and embryo sex was determined (by PCR) on Day 4 (insemination=Day 0). There was no effect (P>0.05) of hormone treatments on rates of cleavage (6 h after cessation of co-incubation with sperm). The only significant effects of added hormones on development were a decrease in the rate of development to at least the morula stage in 165 pg/mL progesterone (0.46+/-0.03 vs. 0.54+/-0.05 in the control, mean+/-S.D.; P<0.05) and a decrease in the blastocyst rate in 0.03 IU/mL FSH (0.34+/-0.00 vs. 0.42+/-0.04 in the control, P<0.05). However, the ratio of male to female embryos was 1.61 and 2.90 following the addition of 99 pg/mL progesterone and 70 pg/mL 17beta-E2, respectively; both of these ratios were different (P<0.01) than in the control group (1.20). In contrast, the addition of FSH to the medium had no significant effect on this ratio (range, 0.78-1.02). We concluded that the addition of progesterone and estradiol to the media during in vitro fertilization did not enhance embryonic development, but significantly increased the proportion of male murine embryos.     

How the FSH/LH ratio and dose numbers in the p-FSH administration treatment regimen, and insemination schedule affect superovulatory response in ewes

We wished to evaluate the effects of FSH/LH ratio and number of doses of p-FSH during a superovulatory treatment on ovulation rate and embryo production (Experiment I). In Experiment II, we studied the efficacy of fertilization after various insemination schedules in superovulated donors. In Experiment I estrus was synchronized in 40 ewes (FGA, for 9 days plus PGF2alpha on Day 7) and the ewes were randomly assigned to four treatment groups as follows (n = 10 ewes each): Group A: four p-FSH doses with the FSH/LH ratio held constant (1.6); Group B: four p-FSH doses with the FSH/LH ratio decreasing (FSH/LH 1.6-1.0-0.6-0.3); Group C: eight p-FSH doses with the FSH/LH ratio held constant (1.6); Group D: eight p-FSH doses and FSH/LH ratio decreasing (1.6-1.6, 1.0-1.0, 0.6-0.6, 0.3-0.3). p-FSH administrations were performed twice daily 12 h apart. The ewes were mated at the onset of estrus and again after 12 and 24 h; then, one ram per four ewes was maintained with the ewes for two additional days. Ovarian response and embryo production were assessed on Day 7 after estrus. Experiment II. Three groups (n = 10 each) of superovulated ewes were inseminated as follows: Group M: mated at onset of estrus; Group AI: artificial insemination 30 h after onset of estrus; M + AI) mating at onset of estrus and intrauterine AI performed 30 h from estrus with fresh semen. Results of Experiment I showed that treatment (D) improved (P < 0.05) ovulatory response in comparison to Groups (C) and (A). The fertilization rate was lower (P < 0.01) in Group D) than Group (A). Also the proportion of transferable embryos was lower in Group (D) in comparison to all the other treatments (P < 0.01). Group A gave the best production of embryos (7.3/ewe; 89.0% transferable). In Experiment II, combined mating plus AI improved fertilization rate (80.3%) compared to both mating (P < 0.01) and AI (P < 0.02) alone.     

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.