The effect of time of intrauterine insemination on the development and viability of embryos collected from superovulated ewes

Eighteen Border Leicester x Scottish Blackface ewes, primed with 300 mg progesterone (12 d) and superovulated with decreasing doses (6, 5, 3 and 2 mg) of porcine FSH, were inseminated with fresh semen, using laparoscopic intrauterine procedures at 48 (Group E) or 60 h (Group L) after exogenous progesterone removal. Five days after insemination, embryos were collected and classified on the basis of their morphological development. During the subsequent 3 d of in vitro culture (38.5 degrees C; 5% CO2) the embryos were evaluated at 24-h intervals. After 72 h, the embryos were individually fixed (24 h) and stained with aceto-orcein and the nuclei were then counted to provide an objective index of cell proliferation and development. Mean (+/-SEM) ovulation rates for the 2 groups (9.2+/-1.5 and 7.1+/-1.2, respectively) and the corresponding percentages (53 vs 59) of embryos collected by laparoscopy were unaffected by insemination time. All donors yielded fertilized ova, but whereas all Group-E donors yielded 1 or more viable embryos (i.e., >32 cells), only 5 Group-L ewes yielded viable embryos (P<0.10). At collection, the percentages of embryos at the morula stage of development were 98 (Group E n = 44) and 39 (Group L n = 38; P<0.001). Few of the remaining ova (Group E = 0% Group L = 8%) were at the 1-cell stage of development when collected, indicating that retarded development post fertilization, not fertilization failure, was the principal consequence of delayed insemination. The percentages of embryos that continued to develop during in vitro culture were 91 and 37 for Groups E and L, respectively (P<0.001), and all of these reached the blastocyst stage. Of these blastocysts, 75 and 50% in Groups E and L hatched in vitro (P<0.10), with mean (+/-SEM) nuclei counts of 148+/-22.7 and 76+/-13.8 (P<0.02), respectively. In conclusion, while delayed intrauterine insemination did not affect the efficiency of ovum collection, it caused a major reduction in the yield of embryos that were capable of developing during in vitro culture. However, fertilization failure accounted for only 13% of the loss in viability following late insemination.     

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.     

Fertilization and ovum recovery rates in superovulated ewes following cervical insemination or laparoscopic intrauterine insemination at different times after progestagen withdrawal and in one or both uterine horns

In Exp. 1, 40 ewes were used in a 2 x 2 factorial design to investigate the effects of intrauterine versus cervical insemination and superovulation using pig FSH or PMSG and GnRH on egg recovery and fertilization rate. Cervical inseminations were carried out at 48 and 60 h (N = 20 ewes) and intrauterine insemination at 52 h (N = 20 ewes) after progestagen pessary withdrawal. Eggs were recovered on Day 3 of the oestrous cycle. Ovulation, egg recovery and fertilization rates were independent of the type of superovulatory hormone used. Fertilization rate was high irrespective of insemination site but intrauterine insemination at 52 h was associated with a significant (P less than 0.01) decrease in egg recovery of over 40% compared with cervically inseminated ewes. In Exp. 2 ewes were inseminated at 36 (N = 5), 48 (N = 6) or 60 (N = 6) h after pessary withdrawal to determine the optimum intrauterine insemination time to maximize both fertilization rate and egg recovery. Egg recovery per ewe flushed was 23, 59 and 67% after intrauterine insemination at 36, 48 and 60 h respectively. Correspondingly, 0, 85 and 100% of the eggs recovered were fertilized. The results of Exps 1 and 2 suggest that when intrauterine insemination occurs before or during ovulation it interferes with oocyte collection by the fimbria. In Exp. 3 egg recovery and fertilization rates were determined after cervical insemination at 48 and 60 h (N = 8) or intrauterine insemination at 48 (N = 9) or 60 (N = 8) h after progestagen withdrawal. Ewes in the last two groups were subdivided and inseminated unilaterally or bilaterally. Egg recovery was high after cervical insemination (95%) but only 36% of these eggs were fertilized. Unilateral intrauterine insemination was as effective as bilateral in ensuring high fertilization rates (100 versus 97%). Intrauterine insemination at 48 h compared with 60 h resulted in a significantly lower (P less than 0.05) percentage of eggs recovered (42 versus 90% respectively). However, reducing the degree of interference by adopting unilateral rather than bilateral insemination did not alleviate the detrimental effects of the 48-h insemination time on egg recovery. From these results we advocate the adoption of intrauterine insemination at 60 h after progestagen withdrawal to maximize fertilization rate and egg recovery in superovulated ewes.     

Dietary-induced suppression of pre-ovulatory progesterone concentrations in superovulated ewes impairs the subsequent in vivo and in vitro development of their ova

In the first of three experiments, eight ovariectomised Greyface ewes primed with exogenous progesterone were used to provide quantitative data on the effects of two contrasting feeding levels (0.3 vs. 1.4 × maintenance) on plasma progesterone concentrations. Over the 9 day study period, mean (± SEM) daily progesterone concentrations were 4.3 ± 0.13 and 3.3 ± 0.17 μg l⁻¹ for the low and high feeding regimens, respectively (P = 0.06), indicating that high feed intake suppressed circulating progesterone levels. The second experiment examined the effect in superovulated Finn-Dorset ewes of a diet supplying either 0.6 (Group L, n = 8) or 2.3 (Group H, n = 8) times their daily energy needs for maintenance, from 1 day before introduction of exogenous progesterone to the time of insemination, on plasma progesterone concentrations and the viability of ova recovered 4 days after insemination. Mean (± SEM) plasma progesterone concentrations were 4.5 ± 0.17 μg l⁻¹ and 2.8 ± 0.16 μg l⁻¹ for L and H ewes, respectively, during the 12 day priming period (P < 0.001). Eight hours after progesterone withdrawal, levels had fallen to 0.9 ± 0.06 μg l⁻¹ and 0.8 ± 0.07 μg l⁻¹, respectively, then rose to 17.8 ± 3.01 μg l⁻¹ and 12.9 ± 2.50 μg l⁻¹ (P > 0.10) at ovum collection. Intervals (mean ± SEM) to oestrous onset (14.5 ± 0.38 h) and the luteinising hormone (LH) surge (27.1 ± 0.98 h) were unaffected by feed intake. Mean (± SEM) ovulation rates (8.1 ± 1.57 vs. 7.8 ± 1.10) and numbers of ova recovered (5.0 ± 1.39 vs. 4.8 ± 1.11) were also similar for each group. However, the proportions of ova considered viable (over 32 cells) at recovery were 0.53 and 0.22 for L and H groups, respectively (P < 0.005). Following 72 h culture (Tissue Culture Medium-199 (M199) + 10% foetal calf serum (FCS)), 0.55 and 0.27, respectively, had developed to blastocysts (P < 0.025). Of ova assessed as viable at recovery, similar proportions (0.86 vs. 0.75) from L and H treatments developed to blastocysts, with corresponding nuclei counts (mean ± SEM) of 55 ± 5.2 and 55 ± 13.2. The third experiment used 12 superovulated Greyface ewes, each offered a different feed level within the range 0.6–2.5 × maintenance, to determine the nature of the relationship between feeding level, pre-ovulatory progesterone concentrations and ovum development at Day 2 following insemination and subsequently during 7 day co-culture (M199 + FCS). Increases in feeding level were accompanied by linear decreases in plasma progesterone (r² = 0.79, P < 0.001), the interval to oestrous onset (r² = 0.52, P < 0.01) and timing of the LH surge (r² = 0.32, P < 0.06). Although undetectable at ovum collection, and somewhat equivocal after 4 day culture, high feeding levels prior to ovulation reduced the proportion of ova (0.16 vs. 0.58) developing to or beyond the expanding blastocyst stage after 7 day culture. Quantitative indices of cell division and protein synthesis confirmed this. In conclusion, excessive feeding during follicular recruitment and oocyte maturation in superovulated ewes imparts a legacy of embryonic loss and developmental retardation.     

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

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

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.     

Ovarian response, ova recovery and fertility in merino ewes superovulated either during the luteal phase of their oestrous cycle or after intravaginal progestagen treatment

Five groups of merino ewes were treated with 1000 i.u. of pregnant mare serum gonadotropin (PMSG) as a single injection per ewe. Three of these groups received treatment on days 7,9 and 11 of their oestrous cycle. Oestrus was synchronized with 125 mg of prostaglandin F2(alpha) (PG) given two days after PMSG. Oestrus in the other two groups was synchronized by intravaginal progesterone sponges inserted for 14 days. In one group, the sponges were inserted nine days after oestrus onset. In the other group the stage of the oestrous cycle was unknown. In both these groups, PMSG was given a day prior to sponge removal. No significant differences were recorded for either the mean numbers of corpora lutea, unovulated follicles or ova recovery between the five groups. However, progestagen synchronized ewes yielded significantly more fertilized ova (p < 0.05) than PG synchronized ewes.     

The effect of embryo quality at freezing on subsequent development of thawed cow embryos

Day 7 to 9 embryos were frozen by a rapid two-step method to ?38°C before being plunged into liquid nitrogen. Glycerol was used as the cryoprotectant and, following thawing, the embryos were cultured for 12 – 24 hours in PBS + 15% heat-treated steer serum. In Experiment 1, embryos were frozen in 2.0 ml glass ampoules or 0.5 ml Cassou straws. Two levels of glycerol (1.0M and 1.4M) gave comparable $\text{in vitro}$ survival rates ($\text{12}\text{20}$ and $\text{13}\text{25}$, respectively). A greater proportion of embryos developed in culture after freezing in straws. In Experiment 2, embryos were classified morphologically before and after freezing into 5 grades (1 = excellent; 2 = good; 3 = fair; 4 = poor; 5 = degenerate). Only embryos of grade 1, 2 and 3 were frozen. The post-thaw survival rates for embryos graded 1, 2 and 3 before freezing were 100% ($\text{11}\text{11}$), 86% ($\text{24}\text{28}$) and 83% ($\text{20}\text{24}$), respectively. Furthermore, the porportion of surviving embryos estimated to be of poor quality (grade 4) was greater for embryos graded 3 before freezing ($\text{13}\text{20}$) than for embryos graded 2 ($\text{6}\text{24}$) or 1 ($\text{1}\text{11}$). The percentage of embryos which developed normally after $\text{in vitro}$ culture for each of the pre-freezing grades 1, 2 and 3 was 91% ($\text{10}\text{11}$), 50% ($\text{14}\text{28}$) and 29% ($\text{7}\text{24}$), respectively. Of the total number of frozen-thawed embryos which developed in culture, $\text{5}\text{31}$ (16%) were of poor quality. The proportion of poor quality developing embryos was greater inembryos graded 3 before freezing ($\text{3}\text{7}$) than those graded 2 ($\text{2}\text{14}$). All of the embryos graded 1 before freezing and which developed in culture were of good quality. Results indicate that, if high post-thaw survival rates are to be obtained, stringent embryo selection processes will be required.     

Location and status of embryos in the genital tract of superovulated cows 4 to 6 days after insemination

Sixty lactating Holstein cows were treated, in 3 groups, with Folltropin and Estrumate to induce superovulation and then bred by artificial insemination (AI). Embryos were collected at slaughter on D 4, 5 or 6 after insemination by flushing separately the oviducts, uterine tip and the remainder of the uterine horn. The embryos and ova recovered accounted for 64.6 ± 4.3% of the ovulations, and there were no differences due to day, side or group. On D 4, 60% of the embryos were found in the oviducts; on Days 5 and 6, 80 and 91%, respectively, were found in the tip of the uterine horn. Viability was independent of the site of recovery; over 91% of the embryos grew and developed in culture for at least 3 d.     

Differences between Belclare and Suffolk ewes in fertilization rate, embryo quality and accessory sperm number after cervical or laparoscopic artificial insemination

Ewe breed has been shown to have a major effect on pregnancy rates following cervical AI using frozen-thawed semen. The main objective of this study was to examine the differences between purebred Belclare and Suffolk ewes (multiparous) in fertilization rate, number of accessory sperm and stage of embryo development on day 6 after cervical or laparoscopic AI with frozen-thawed semen. In experiment 1, Belclare and Suffolk ewes were synchronized for 12 days and were either cervically inseminated (year 1: n=28 and 31; year 2: n=16 and 15, respectively) or laparoscopically inseminated (year 2: n=13 and 14). In experiment 2, superovulated Belclare (n=4) and Suffolk (n=13) ewes were laparoscopically inseminated. All ewes were slaughtered 6 days after AI; oocytes/embryos were recovered, morphologically graded and stained to assess the number of cells and accessory spermatozoa. Data from both experiments were combined for statistical analysis. The proportion of ewes with fertilized oocytes was significantly higher following laparoscopic AI compared with cervical AI (54% versus 19%). More Belclare than Suffolk ewes yielded fertilized oocyte(s) after cervical AI (34% versus 10%, P<0.02) but there was no difference after laparoscopic AI (62% versus 60%). From the ewes that yielded at least one fertilized oocyte the proportion of Belclare ewes with embryos at the morula/blastocyst stage was significantly greater than for Suffolk ewes (94% versus 59%, P<0.02). A higher proportion of Belclare than Suffolk ewes had evidence of sperm reaching the site of fertilization following cervical AI (39% versus 15%, P<0.02) but there was no difference after laparoscopic AI (62% versus 64%, P>0.8). Amongst the ewes with evidence of sperm at the site of fertilization, laparoscopic AI resulted in a higher number of sperm per oocyte/embryo or per ewe than cervical AI (P<0.01). These results suggested that the difference in pregnancy rate between Suffolk and Belclare ewes following cervical AI was due to: (i) sperm traversing the cervix and uterus in a higher proportion of Belclare than Suffolk ewes, leading to a higher incidence of fertilization and (ii) the lower developmental competence of fertilized oocytes from Suffolk ewes.     

In vitro evaluation of early embryo viability and development in summer heat-stressed, superovulated dairy cows

Our study evaluated the viability and the development in vitro of embryos flushed from superovulated heat-stressed (hot season) and unstressed (cool season) Holstein cows 6 to 8 d after artificial insemination (AI). Plasma progesterone (P(4)) levels were measured in all cows. The incidence of unfertilized ova was significantly increased in hot-season cows (P < 0.05). There was no seasonal effect on the highly variable P(4) levels 6 to 8 d after AI. Morulae and blastocysts flushed from hot-season cows were significantly less viable in culture than morulae or blastocysts flushed from cool-season cows (P < 0.001 and P < 0.0025, respectively). Furthermore, there was a significant seasonal effect both on blastulation (P < 0.0025) and hatching of blastocysts developed from morulae in culture (P < 0.005) and on expansion of blastocysts placed in culture at the blastocyst stage (P < 0.05). These data lead us to suggest that the reduced fertility of summer heat-stressed dairy cows may result from decreased viability and developmental capacity of Day-6 to Day-8 embryos. In addition, the results indicate that the efficiency of embryo transfer procedures may be significantly lowered by the reduced embryo viability associated with hot weather and that reduced embryo viability may be the cause of the well-documented seasonal reduction in the efficiency of AI.     

A serum-free, cell-free culture system for development of bovine one-cell embryos up to blastocyst stage with improved viability

With the aim of developing a serum-free, cell-free culture system for embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 with the following supplements: 1) BSA alone (10 mg/ml); 2) BSA with ITS (5 mug/ml insulin, 5 mug/ml transferrin and 5 ng/ml selenium; BSAITS medium); 3) estrous cow serum alone (ECS; 10%); or 4) ECS with BOEC (bovine oviduct epithelial cells) (Experiment 1). In Experiment 2, embryos were cultured in BSAITS medium with or without feeding with fresh medium on Day 4 (day of insemination = Day 0). Embryos were evaluated on Day 2 for first cleavage, on Day 7 for morulae and blastocysts, and on Day 8 for blastocysts. Blastocysts from Experiment 1 were frozen in 10% glycerol in PBS, thawed and further cultured in ECS medium with BOEC for 48 h, and evaluated for formation of a distinct blastocoel, or expansion and hatching of blastocysts. In vivo-developed, Grade-1 and Grade-2, 7-d-old embryos served as control for the freezing, thawing and subsequent culture procedures. The percentage of first cleavage did not differ between the treatments (74 to 79% in Experiment 1 and 80 to 83% in Experiment 2). The percentage of blastocysts developed in BSAITS medium did not differ from that in ECS medium whether BOEC were present or not. However, medium with BSA alone had fewer blastocysts than any other culture system (P<0.05). Feeding embryos with fresh BSAITS medium on Day 4 did not lead to any further increase in the proportion of blastocysts. The culture systems had a significant effect on the post-thaw viability of blastocysts developed in them (P<0.001). Blastocysts developed in BSAITS medium had better (P<0.05) viability (14/38) than those from medium with ECS alone (1/27) or with ECS and BOEC (3/37). The post-thaw survival of control embryos was 80% (n=30). One of the three transfers of BSAITS-treated, frozen-thawed blastocysts resulted in a pregnancy. The results indicate that a serum-free, cell-free culture system can support the development of IVM-IVF bovine oocytes up to the blastocyst stage with better viability than a complex co-culture system.     

Effect of variation in embryo stage on the establishment of pregnancy, and embryo survival and growth in ewes with two embryos

Embryos at different stages of development were transferred to recipient ewes on Day 6 to investigate the effect of variation in stage of development on embryo survival and growth. Three groups of ewes received 2 embryos that were at the same stage of development, Day 4, Day 6 or Day 8. A fourth group received 1 Day-4 and 1 Day-8 embryo. At autopsy on recipient Day 34 there were no significant differences in embryo survival (Day 4, 34%; Day 6, 50%; Day 8, 46%; and Day 4 and 8, 48%). Fetuses developing from Day-8 embryos were heavier than others (Day 4, 1.10 +/- 0.06 g; Day 6, 1.15 +/- 0.06 g; Day 8, 1.41 +/- 0.08 g; P less than 0.05). In Group 4 neither survival nor growth of embryos was significantly affected by the presence of an embryo at a different stage of development. The ability of the uterus to stimulate development of a relatively retarded embryo is confirmed. Apparently the uterus has less effect in slowing the development of advanced embryos.     

Effects of caffeine and/or heparin in a chemically defined medium with or without glucose on in vitro penetration of bovine oocytes and their subsequent development

Bovine cumulus-enclosed oocytes were matured in culture for 22 to 24 h, freed from cumulus cells and inseminated with frozen-thawed spermatozoa in a chemically defined medium containing 1 mg polyvinylalcohol/ml with or without 5 mM caffeine and/or 10 micrograms heparin/ml and 13.9 mM glucose. Penetration of oocytes was observed only in the medium containing caffeine and/or heparin. Regardless of the presence of glucose, similar proportions of oocytes were penetrated in the medium containing heparin with (73 and 83%) or without (36 and 41%) caffeine. However, when the medium was supplemented with caffeine only, a higher penetration rate was observed in the presence (41%) than in the absence (27%) of glucose. When oocytes inseminated in medium containing caffeine and heparin with or without glucose were cultured in a chemically defined, protein-free medium, 72 and 90% and 9 and 21% of inseminated oocytes developed to the > or = 2-cell and blastocyst stages 48 and 192 h post insemination, respectively. These results, obtained using chemically defined conditions, indicate that glucose is required for stimulating fertilization in vitro of bovine oocytes and that synergistic action of caffeine and heparin appears independently of the reversing activity of caffeine on the inhibition of heparin-induced sperm capacitation by glucose.     

The effect of freezer type, cryoprotectant, and processing methods on viability of frozen embryos

Two hundred forty excellent-quality blastocysts flushed from 53 superovulated Holstein heifers were frozen by 1 of 16 procedures in a 2 x 2 x 2 x 2 factorial design. The main effects included a simple, inexpensive, portable mechanical freezing unit instead of a programmable Liquid Nitrogen (LN) freezer for freezing bovine embryos, cryoprotective agents dimethyl sulfoxide (DMSO) and glycerol, addition rates of the cryoprotectants and freezing rates. Embryo viability was assessed morphologically and by fluorescein diacetate (FDA) evaluation. Neither the type of freezer, the cryoprotectant nor the rate of cryoprotectant addition affected embryo viability. Embryo survival after 12 h of incubation was higher (P < 0.05) using a conventional freezing rate than a two-step method (37.2 vs 16.5%). The correlation coefficient between viability evaluation methods (morphology vs FDA) was influenced by cryoprotectant and embryo processing methods and ranged from -0.13 to +0.70. This study indicates that more simplified embryo freezing equipment and handling procedures may provide protection equal to that of more complicated, expensive equipment and more time-consuming methods. Economical on-farm embryo freezing is feasible.     

Effects of cadmium on preimplantation mouse embryos in vitro with special reference to their implantation capacity and subsequent development

A short exposure to 5 or 10 micrograms/ml cadmium chloride for 24 hours disturbed the in vitro development of four-cell and morula-stage embryos of F1 (C57 female X A2G male) mice. Morulae appeared to be less sensitive to cadmium than four-cell embryos. However, the in vitro development of four-cell embryos through compaction to morulae was not affected, though most treated embryos degenerated and decompacted later. It was proposed that cadmium toxicity may not be acting through contact effects on the cell surface and cytoskeleton. It probably interferes with the general energy metabolism of the cells. Although 1 microgram/ml cadmium did not disturb the subsequent in vitro development beyond the implantation stage, a reduced capacity of implantation in vivo was observed after surgical transfer to recipients. In spite of the effects of cadmium salts on the maternal side, the present results suggest that a direct embryotoxic and teratogenic activity of cadmium cannot be excluded.     

The development of Xenopus tropicalis transgenic lines and their use in studying lens developmental timing in living embryos

The generation of reporter lines for observing lens differentiation in vivo demonstrates a new strategy for embryological manipulation and allows us to address a long-standing question concerning the timing of the onset of differentiation. Xenopus tropicalis was used to make GFP reporter lines with (gamma)1-crystallin promoter elements directing GFP expression within the early lens. X. tropicalis is a close relative of X. laevis that shares the same ease of tissue manipulation with the added benefits of a diploid genome and faster life cycle. The efficiency of the Xenopus transgenic technique was improved in order to generate greater numbers of normal, adult transgenic animals and to facilitate in vivo analysis of the crystallin promoter. This transgene is transmitted through the germline, providing an accurate and consistent way to monitor lens differentiation. This line permitted us to distinguish models for how the onset of differentiation is controlled: by a process intrinsic to differentiating tissue or one dependent on external cues. This experiment would not have been feasible without the sensitivity and accuracy provided by the in vivo reporter. We find that, in specified lens ectoderm transplanted from neural tube stage donors to younger neural-plate-stage hosts, the onset of differentiation, as measured by expression of the crystallin/GFP transgene, is delayed by an average of 4.4 hours. When specified lens ectoderm is explanted into culture, the delay was an average of 16.3 hours relative to control embryos. These data suggest that the onset of differentiation in specified ectoderm can be altered by the environment and imply that this onset is normally controlled by external cues rather than by an intrinsic mechanism.     

Fertility of ewes following artificial insemination with semen frozen in pellets or straws, a preliminary report

In two trials involving the artificial insemination of 194 ewes, the fertility of ram semen was examined following freezing, either in pellet form or in straws, and after storage in a chilled state (15 degrees C) for up to 16 hours. Estrus was synchronized in ewes by intravaginal sponge (MAP) treatment for 14 days. At sponge removal 600 IU PMSG was injected and the ewes received two inseminations 50 and 60 hours later. Fertility was assessed at lambing. In trial 1, the mean lambing rate of 52% (16 31 ) for semen frozen in pellets was higher than 29% (9 31 ) for semen frozen in straws but this difference was not significant. In trial 2, ewes inseminated with chilled semen and semen frozen in pellets had lambing rates of 83% (44 53 ) and 55% (44 79 ) respectively (P<0.001).     

The effect of GnRH,eCG and progestin type on estrous synchronization following laparoscopic AI in ewes

The aim of the experiment was to evaluate the effects of GnRH and/or eCG and progestin type (implant versus CIDR) on the induction of estrus and pregnancy rate following laparoscopic AI (LAI) with frozen semen. In the first trial, ewes (n = 129) were treated with norgestomet implants for 14 days. At implant removal ewes received eCG (400 IU) and/or GnRH (25 μg) 36 h after removal, resulting in control, eCG, GnRH, and eCG/GnRH groups (n = 30–34/group). In trial 2, ewes (n = 36) were treated with intravaginal fluorogestone acetate sponges (FGA) or CIDR for 12 days. After withdrawal, half of the ewes from each progestin group received eCG (400 IU), resulting in sponge, sponge/eCG, CIDR and CIDR/eCG groups (n = 8–10/group). In both trials, estrous activity was assessed using a vasectomized ram from the time of progestin removal to laparoscopic AI with frozen semen 58–60 h (trial 1) or 54–56 h (trial 2) following cessation of treatment. In trial 1, GnRH decreased (P < 0.05) the percentage of ewes in estrus (GnRH, 75.8% versus control, 93.8% versus eCG/GnRH, 94.1%), however pregnancy rates were similar in all groups (control, 53.1%; eCG, 70.0%; GnRH, 51.5%; eCG/GnRH, 55.9%, respectively). In trial 2, neither the type of progestin nor eCG treatment effected the percentage of ewes in estrus (sponge, 75.0%; sponge/eCG, 100.0%; CIDR, 100.0%; CIDR/eCG, 90.0%). However, pregnancy rates following LAI were higher (P < 0.05) when ewes were treated with eCG (progestin + eCG, 73.7% versus progestin alone, 41.2%). Results demonstrate that the source of progestin does not influence the expression of estrus or the proportion of ewes pregnant following LAI. When progestin treatment protocols are used in combination with eCG, pregnancy rates can be increased. A dose of GnRH near the end of progestin treatment may decrease the estrous response, by inducing ovulation before normal expression of estrus.