Nonsurgical Embryo Collection Research Articles

The presence of estradiol benzoate in the cervical relaxation treatment for non-surgical embryo collection does not impair embryonic morphological quality, cryosurvival, and gene expression profile

Non-surgical embryo recovery (NSER) is usually preceded by a cervical relaxation in ovine donors, based on estradiol benzoate (EB), prostaglandin (PGF), and oxytocin (OT). However, it is hypothesized that, due to poorly understood mechanisms, EB can result in embryotoxic actions. To evaluate this, 20 min before NSER superovulated sheep were induced to cervical relaxation with 0.0 (G0.0), 0.5 (G0.5), or 1.0 mg (G1.0) of EB associated with 37.5 μg of PGF 16 h before NSER and 50 IU of OT. In doing so, the efficiency and duration of the NSER procedure showed no compromise (P > 0.05). Additionally, the presence of EB did not affect (P > 0.05) the embryo's morphological quality, the development dynamics, or the abundance of transcripts associated with embryonic quality (OCT4 and NANOG), cellular stress (HSP90 and PRDX1), and apoptosis (BCL2 and BAX). A similar result (P > 0.05) was also observed when comparing embryonic cryosurvival at 24 (52.0, 52.0, and 54.0) and 48 h (60.0, 54.0, and 58.0) of in vitro culture (G0.0, G0.5, and G1.0, respectively). Thus, we can conclude that EB use does not compromise embryonic quality and cryoresistance.

Polymorphisms within the <em>IGF1</em> and <em>IGF1R</em> genes associated with superovulation-related traits in Holstein dairy cows managed in a semiarid environment

Embryo transfer (ET) is an artificial reproductive technology used for the genetic improvement of cattle. High variation has been observed in superovulation (SPO) and embryo flush recovery, which appear to be influenced by donor cow genetics. Then, the objective was to identify single nucleotide polymorphisms (SNPs) from the genes IGF1 and IGF1R associated with reproductive traits related to SPO response in dairy cows raised in a semiarid region. Sixty-four Holstein cows were subjected to SPO, artificial insemination, and nonsurgical embryo collection. Individual blood samples were collected and used to genotype 13 SNPs from the genes IGF1 and IGF1R. Additional blood samples were collected to measure anti-Mullerian hormone (AMH) concentrations. Ovarian and embryo traits related to SPO response were evaluated. A mixed-effects model was used to identify associations between SNPs and SPO-related traits. A regression model was implemented to calculate allele substitution effects. From 13 SNPs, the SNP rs109763947 in the gene IGF1 and the SNPs rs110343126 and rs208140993 in the gene IGF1R were predictors for six traits evaluated in superovulated cows. The most favorable genotypes for these SNPs were CC, AA and GG, respectively. A linear trend was detected, suggesting an additive effect of the genes. Moreover, all traits evaluated in the current study improved as the number of favorable SNP genotypes increased, confirming a positive contribution of the SNP genes. In conclusion, three SNPs in the genes IGF1 and IGF1R were marker predictors for reproductive traits related to SPO response and embryo production in Holstein cows managed in a semiarid region.

CAPABILITY OF DIFFERENT BREEDS OF DONOR CATTLE TO PRODUCE EMBRYOS AT THE CIPELANG LIVESTOCK EMBRYO CENTER, BOGOR, WEST JAVA

The objective of this research was to investigate the capability of the donor cattle to produce embryos by comparing three cattle breeds, namely Bos taurus beef cattle (Limousin), Bos taurus dairy cattle (Friesian Holstein/FH), and Bos indicus beef cattle (Peranakan Ongole/PO). This research was observational method with in vivo embryo production method and a nonsurgical embryo collection method (flushing). The data collected were then processed using the one way ANOVA method. The results showed that the number of CL, response rate, total number of embryos, and recovery rate in PO cattle, FH cattle, and Limousin cattle did not differ significantly (P0.05). The appropriate number of embryos to be transferred and degenerative embryo were significantly different among PO cattle, FH cattle, and Limousin cattle (P0.05), while the unfertil ova, fertilization rate, and transferable embryo did not differ among cattle breed (P0.05). This research concluded that each breed of the donor cattle produced embryos with varies quality and the best quality of embryo production is found in Limousin cattle.

LH pattern and ovarian response in ovsynch-synchronized superovulated goats induced to ovulate with GnRH or hCG

The intention of the present investigation was to compare a GnRH agonist and hCG as ovulation inducing agents to apply fixed-time insemination in ovsynch synchronized superovulated goats. Three groups of 17 does of the Boer goat breed were synchronized, superovulated and ovulation induced with a GnRH analog, hCG or physiological saline, followed by mating and non-surgical embryo collection. Part of the does were provided with subcutaneous progesterone implants after mating to compensate for frequently occurring premature corpus luteum regression. Estrus detection was conducted with aproned bucks, ovarian function was monitored sonographically and by progesterone analyses and blood samples were drawn to generate LH profiles. Practically all does responded to treatment and occurrence and duration of estrus as well as the ovulatory response were unaffected by treatment. The LH surge provoked by administration of the GnRH analog buserelin emerged within 1.0 h and was closely synchronized, significantly more so than after administration of hCG or physiological saline, commencing after 11.8 and 14.9 h, respectively, with no significant differences amongst each other. It would seem that hCG is not effective in bringing about a LH surge. The interval between commencement of the LH surge and ovulation was, across treatment groups, 24.2 h with no significant difference among groups. Ovulatory response and yield of transferable embryos obtained by nonsurgical collection were modest with no significant differences among groups.

Predictive markers for superovulation response and embryo production in beef cattle managed in northwest Mexico are influenced by climate

Important components of a successful embryo transfer program in cattle are the superovulatory response and embryo production. Anti-Müllerian hormone (AMH) and antral follicle count (AFC) have been proposed as biomarkers of the reproductive response in superovulated cattle; however, their predictive ability appears to be influenced by climate. Objective herein was to evaluate AMH and AFC as predictors of the number of ovarian follicles, corpora lutea, collected embryos and transferable embryos in cows managed in northwest Mexico under different climates. Multiparous Simmental beef donor cows were assigned in two experimental climatic groups, cool (January 15– May 14; n=66) and warm (May 15– September 14; n=62). All cows were given a superovulatory treatment, and then subjected to artificial insemination and non-surgical embryo collection. Transrectal ultrasonography was performed to determine AFC. Blood samples were collected to measure blood AMH concentrations. A mixed effects model was used to analyze superovulatory and embryo production responses in cattle including AMH, AFC and climate as fixed effects, age as covariate and herd as random effect. All fixed effects resulted as significant (P<0.05) sources of variation for the evaluated traits, and interactions between AMH/AFC with climate were observed but only for transferable embryos. Pearson correlations were calculated between AMH/AFC with superovulatory and embryo production variables for cool and warm experimental groups, and a regression analyses was performed to quantify these relationships. Cows with either AMH>400 pg/mL or intermediate to higher AFC increased (P<0.05) mean values for superovulatory and embryo production variables, suggesting both AMH and AFC as predictors. Climate also influenced cow responses as number of follicles, corpora lutea, as well as collected and transferable embryos increased 33, 38, 40 and 35%, respectively, during cool- (8-23 °C) compared to warm-climate (24-34 °C; P<0.05). Both AMH and AFC were moderately correlated with number of follicles, corpora lutea and collected embryos (P<0.05) and highly correlated with transferable embryos (P<0.01) during cool climate; conversely, these correlations were low (P>0.05) for donor cows managed in the warm months. Regression analyses suggested that total number of transferable embryos increased 0.005 per 1 pg/mL of AMH and improved 0.484 per 1 unit of AFC. In conclusion, superovulatory and embryo production responses in Simmental beef cows appear to be closely associated with AMH and AFC, which are proposed as predictors for transferable embryos; however, such relationships were only detectable in absence of heat stress.

Superovulation of Boer goats with different synchronization regimens at different times of the year in the northern temperate zone

The present study addresses a comparison of various estrous cycle control protocols and the effect of season in the northern temperate zone on the superovulatory response in Boer goats. Four groups of 12 does each were treated in February, May, August and November, respectively. Does were provided with progestogen containing intravaginal pessaries to synchronize estrus. Half the does of each group received sponges impregnated with 20 mg fluorogestone acetate (“Cronolone”), the other half “Eazy-Breed” CIDRs containing 0.3 mg progesterone. Pessaries remained in place for 7 days. Within each subgroup half the does were treated with PGF2α (“Dinoprost”) at pessary insertion, the other half at pessary withdrawal. Beginning 48 h before pessaries were removed does received i.m. injections of 4, 4, 2, 2, 2 and 2 Armour Units (AU) FSH, supplemented with 40% LH, at 12 h intervals. Estrus detection was conducted at 8 h intervals and ovarian activity was monitored by daily transrectal ultrasonography. Does were mated and 7 days later non-surgical embryo collection was conducted. It transpired that the type of intravaginal pessary had no effect on the time passing between pessary withdrawal and onset of estrus (sponge: 40.7 h vs. CIDR: 35.2 h), number of ovulations/doe assessed by echographic counting of collapsed large follicles (7.2 vs. 7.6) or corpora lutea (7.1 vs. 9.8), embryo recovery rate (39% vs. 41%) and proportion of transferable embryos (66% vs. 65%). The number of collapsed large follicles was significantly higher than that of corpora lutea on the day before embryo collection (7.4 vs. 8.5; P < 0.05), the correlation between the two being r = 0.55 (P < 0.05). The effect of PGF2α administration at the onset or at the end of progestogen treatment and the effect of season were not significant, except for a shorter interval between pessary removal and onset of estrus in August in comparison to the other months (29.3 vs. 39.4, 40.6 and 42.1 h; P < 0.05). In conclusion, Boer goats, although being seasonal breeders in the northern temperate zone, will respond to superovulatory treatment and produce embryos of high quality year round regardless of type of pessary and time of PGF2α administration.

Combined treatment with oestradiol benzoate, d-cloprostenol and oxytocin permits cervical dilation and nonsurgical embryo recovery in ewes.

This study examined the feasibility of transcervical embryo recovery after the hormonal treatment to induce cervical dilation, following the 7-day oestrous synchronization protocol in multiparous Santa Inês ewes. A total of 23 cyclic ewes received two doses of 37.5μg of d-cloprostenol by latero-vulvar route 7days apart. After the second injection of d-cloprostenol, the ewes were checked for oestrus (every 12hr) and then mated by fertile rams throughout the oestrous period. All ewes received 37.5μg of d-cloprostenol (latero-vulvar) and 1mg of oestradiol benzoate by either intramuscular (EBim group; n=12) or intravaginal (EBivg group; n=11) route 16hr before embryo flushing. Twenty minutes before the flushing, 50IU of oxytocin were administered intravenously. The oestrous response (i.e., the percentage of ewes that showed signs of oestrous behaviour after the second d-cloprostenol injection) was 91.3% (21/23). The proportion of successfully penetrated ewes (81.8% compared with 80.0%), the mean duration of embryo flushing (24.7±2.0min compared 26.2±1.9min), the flushing fluid recovery rate (94.8±1.3% compared with 91.0±2.9%) and the average number of structures recovered per ewe (0.5±0.4 compared with 0.8±0.4) did not vary (p>0.05) between the EBim and EBivg groups. Viable embryos were recovered from 41.2% (7/17) of successfully penetrated ewes. It can be concluded that nonsurgical (i.e., transcervical) embryo collection can be performed in oestrous-synchronized Santa Inês ewes pretreated with d-cloprostenol, oxytocin and oestradiol benzoate, with the latter hormone administered by either the intramuscular or intravaginal route.

Effect of repeated dual superovulation using FSH and PMSG+FSH on ovulatory response in Murrah buffalo

The current experiment was conducted to determine the ovulatory response of Murrah buffaloes subjected to repeated therapeutic doses of FSH and PMSG+FSH in two successive periods. Three elite, multiparous, Murrah buffaloes, 80 to 120 days postpartum, were included in multiple-ovulation embryo transfer (MOET) schedule. The estrus was synchronized with one injection of prostaglandin. Superovulatory treatment was started from day 10 of induced estrus after ablation of dominant follicle of over 10 mm diameter, using 5.0 MHz convex-array intravaginal transducer using a B-mode scanner with aspiration assembly. Study was undertaken in two consecutive periods so each animal was treated with PMSG+FSH in first superovulation period and FSH on the Second period after short interval period (14 day cycle). For FSH treated, Folltropin was administered in a twice-daily descending dose schedule (0–5, 5–4, 4–3, 3–2, 2–2 ml; 20 mg/ml, total dose 600 mg of FSH. For PMSG+FSH treated, an injection of PMSG (1000 IU) was given in the evening of day 10 followed by dose 400 mg FSH and schedule as of FSH treated group from the evening of day 11. All donors received prostaglandin injection on day 13 in morning and evening. Donors were inseminated with frozen thawed semen of proven bull on Day 15 a.m. and p.m. and Day 16 a.m. Nonsurgical embryo collection was carried out on Day 5 post-insemination. There were no differences between the mean numbers of follicles in the same buffaloes treated with PMSG+FSH or FSH at any time throughout the experimental period. At AI, similar numbers of corpora lutea were observed for PMSG+FSH and FSH. On the day of flushing, almost similar mean numbers of corpora lutea counted in PMSG+FSH and FSH treated buffaloes In conclusion, ovarian response in Murrah buffaloes treated with PMSG+FSH or FSH for two superovulatory periods were nonsignificantly different.

212 EFFECT OF A LOW DOSE OF eCG ON SUPEROVULATION AND EMBRYO COLLECTION IN WOOD BISON DURING THE BREEDING SEASON

In an effort to conserve a threatened Canadian species, Bison bison athabascae, we developed an embryo collection protocol subsequent to superovulatory treatment involving two doses of FSH diluted in hyaluronan given 48 h apart. The follicular response to superstimulatory treatment was satisfactory, but many follicles did not ovulate, thus limiting the number of embryos collected. Based on recent results in cattle, where replacement of the final doses of FSH with a low dose of eCG resulted in the recovery of a greater number of ova/embryos, the objective of the present study was to evaluate the effect of adding eCG to the superovulatory protocol to increase ovulation rate, embryo collection, and embryo quality in wood bison during the breeding season (September). Ovarian synchronization was induced in wood bison (n = 24) by treatment with a luteolytic dose of prostaglandin (500 mcg, Cloprostenol) followed 8 days later by transvaginal ultrasound-guided follicular ablation. Follicular wave emergence (Day 0) was defined as the day after follicle ablation. Bison were assigned randomly to two groups: FSH (n = 12) and FSH+eCG (n = 12). FSH was diluted in hyaluronan (5 mg mL–1, MAP-5, Bioniche Animal Health, Belleville, ON, Canada) and given intramuscularly on Day 0 (300 mg) and Day 2 (100 mg) in both groups. Bison in the FSH+eCG group received 450 IU eCG (Pregnecol, Bioniche Animal Health) intramuscularly on Day 3, and bison in both groups were administered a luteolytic dose of prostaglandin on Day 3. On Day 5, bison were given 2500 IU hCG (Chorulon, Merck Animal Health, Summit, NJ, USA) intramuscularly to induce ovulation. The bison were artificially inseminated with chilled semen 12 and 24 h after hCG treatment. Nonsurgical embryo collection was performed on Day 13. The ovaries were examined by transrectal ultrasonography on Days 5, 7, and 13 to record the follicular response, ovulation rate, and number of corpora lutea (CL), respectively. Results were compared between groups by t-test or chi-square test (Table 1). The number of ovulatory-sized follicles (≥9 mm), ovulation rate (number ovulations/ovulatory-sized follicles), number of CL, number of ova/embryos, and number of transferable embryos were not different between groups (P = 0.23, P = 0.19, P = 0.25, P = 0.18, P = 0.09, respectively). In conclusion, the superovulatory response and embryo collection rate in wood bison approached that observed in cattle, but were not improved by the addition of a low dose of eCG. Table 1.Response of wood bison (mean ± s.e.m.) to superovulatory treatment with or without eCG

Ultrasound-guided non-surgical embryo collection in the common marmoset

Experimental primate embryology has been hampered by limited access to embryos. In addition to surgical techniques, the less stressful non-surgical technique of uterine flushing has been developed but has had only limitedly used in recovering pre-implantation embryos from marmoset monkeys. In this study, we introduce the use of ultrasonography during marmoset non-surgical uterine flushing to make the cannulation easier, to further reduce stress, and to ensure thorough uterine flushing. We were able to cannulate in 99% of the transcervical cannulation attempts, repeat the flushing up to 17 times with the same animal, and recover up to 90% of the ovulation products. We also found that 8-cell or earlier stage embryos could be frequently obtained by non-surgical uterine flushing at 4 or 5 days after ovulation. The easiness and effectiveness of this novel ultrasound-guided technique will enable more research groups to study marmoset embryology and facilitate progress in this field.

Superovulatory Response Following Transvaginal Follicle Ablation in Murrah Buffalo: Effect of FSH or PMSG+FSH

The current experiment was conducted to reduce the cost of superovulation by reducing the dose of FSH by partial replacement with PMSG. Five elite, multiparous, Murrah buffaloes, 80 to 120 days postpartum, were included in multiple-ovulation embryo transfer (MOET) schedule. The estrus was synchronized with one injection of prostaglandin. Superovulatory treatment was started from day 10 of induced estrus after ablation of dominant follicle of over 10 mm diameter, using 5.0 MHz convex-array intravaginal transducer using a B-mode scanner with aspiration assembly. Study was undertaken in two groups. For FSH treated group, Folltropin was administered in a twice- daily descending dose schedule (0–5, 5–4, 4– 3, 3–2, 2–2 ml; 20 mg/ml, total dose 600 mg of FSH. For PMSG+FSH treated group, an injection of PMSG (1000 IU) was given in the evening of day 10 followed by dose 400 mg FSH and schedule as of FSH treated group from the evening of day 11. All donors received prostaglandin injection on day 13 in morning and evening. Donors were inseminated with frozen thawed semen of proven bull on Day 15 a.m. and p.m. and Day 16 a.m. Nonsurgical embryo collection was carried out on Day 5 post-insemination. There were no differences between the mean numbers of follicles in buffaloes treated with FSH or PMSG+FSH at any time throughout the experimental period. At AI, similar numbers of corpora lutea were observed for PMSG and FSH, respectively. On the day of embryo recovery, almost similar mean numbers of corpora lutea counted in FSH and PMSG+FSH treated buffaloes were 5.5+2.5 and 2.6+0.33, respectively. A significant increase in number of anovulatory follicles in FSH and PMSG+FSH (10+0.0; 9.3+0.3, respectively) was observed, which may be the reason for lower total embryo recovery and viable embryo recovery. In conclusion, superovulatory response of PMSG+FSH was similar to FSH treated group and can be used to reduce the cost of superovulation in MOET program.

310 DOES PROGESTERONE OR EQUINE CHORIONIC GONADOTROPIN IMPROVE THE SUPEROVULATORY RESPONSE AND EMBRYO QUALITY IN WOOD BISON DURING THE ANOVULATORY SEASON?

Superovulation protocols are being developed in wood bison (Bison bison athabascae), a threatened Canadian species. In initial studies, 2 doses of FSH diluted in hyaluronan given 48 h apart were successful for inducing ovarian superstimulation in wood bison, and ovulation rate was improved by final treatment with hCG instead of LH (Palomino et al. 2012 Reprod. Fertil. Dev. 24, 226). In that study, exogenous progesterone had no effect on the number of ovulations, but embryo quality could not be evaluated because of the low number of embryos collected. In beef cattle, replacement of the final doses of FSH with eCG has resulted in the recovery of a greater number of ova/embryos. The objective of the present study was to evaluate the effect of exogenous progesterone (PRID) on embryo quality and to determine if the addition of eCG increases the ovulation rate in superstimulated wood bison. Follicular ablation was done to synchronize follicular wave emergence in 26 wood bison cows during the anovulatory season (May). Cows were assigned randomly to 3 groups: PRID/no eCG (n = 8), PRID/eCG (n = 9), and no PRID/eCG (n = 9). A PRID was inserted on the day of follicular ablation (Day –1) in respective groups. In all bison, FSH diluted in hyaluronan (5 mg mL–1, MAP-5, Bioniche Animal Health Inc., Belleville, Ontario, Canada) was given intramuscularly on Day 0 (300 mg) and Day 2 (100 mg). On Day 3, a single dose of 450 IU of eCG (Pregnecol, Bioniche Animal Health Inc.) was given intramuscularly and the PRID were removed in the corresponding groups. On Day 5, all bison were given 2500 IU of hCG (Chorulon, Merck, Whitehouse Station, NJ, USA) intramuscularly to induce ovulation. Bison were inseminated with chilled semen 12 and 24 h later. Nonsurgical embryo collection was performed on Day 14. Transrectal ultrasonography was done to record the ovarian response, ovulation rate, and number of corpora lutea (CL). Results were compared by ANOVA and Chi-squared test (Table 1). The number of ovulatory-sized follicles (≥9 mm) on Day 5 did not differ among groups. Ovulation rate was lowest in bison treated with both a PRID and eCG (P &lt; 0.05). There were no differences among groups in the number of CL on Day 14, the number of ova/embryos collected, or the number of transferable embryos. The superovulatory response and embryo collection rate in the present study were higher than in any previous reports in bison. The ovulation rate was not improved by the addition of eCG treatment, and exogenous progesterone had no effect on embryo quality. Table 1.Response of superstimulated wood bison (mean ± SEM) to treatment with exogenous progesterone (PRID) and/or eCG

155 CHARACTERIZATION OF LIPID DROPLETS IN BOS INDICUS AND BOS TAURUS EMBRYOS

Accurate evaluation of bovine embryos for assessing developmental stage and quality is critical to the success of any embryo transfer program. However, this evaluation process has been reported to be highly subjective in Bos indicus (BI) and can vary as much as 23% compared with that of Bos taurus (BT). These differences in assessment may be related to the quantity of lipid droplets (LD) within the embryo, which has been shown to have a negative effect in cryopreserving embryos. The aim of the present study was to characterize the number and size of LD in different developmental stages of fresh embryos from BI and BT and to compare LD across the three different embryo quality grades (1 = excellent or good, 2 = fair, and 3 = poor). Nonsurgical embryo collection was performed 7 days post-insemination in 10 BI and 10 BT females. Forty-eight embryos were evaluated for stage and grade using stereoscopic microscopy, processed for transmission electron microscopy, and stained with Nile red. Digitalized images were analyzed with ImageJ (National Institutes of Health, Bethesda, MD, USA), contour of lipid droplets were designed, and values of perimeter, area, and fluorescence intensity were assessed. Nonparametric statistical analysis (Mann–Whitney) was utilized. There was no difference in LD number for BT or BI for morulae and blastocyst; however, BI morulae presented larger LD compared with blastocyst stage embryos (286 µm2 v. 223 µm2; P &lt; 0.05). Likewise, BI TF cells had more LD compared with inner cell mass (ICM) cells (48 v. 36; P &lt; 0.05). BT TF cells exhibited larger LD compared with ICM cells (149 µm2 v. 128 µm2; P &lt; 0.05), while BI embryos exhibited a larger area of LD in the ICM compared with the TF (591 µm2 v. 472 µm2; P &lt; 0.05). In all embryos, BI contained more lipid droplets than BT (78 v. 49; P &lt; 0.05). Across all quality grades (good, fair, and poor) there was no difference in the number of LD in BT embryos; however, BI grade-3 embryos presented more LD than grade-1 (36 v. 25). BT embryos LD were larger than BI LD (907 µm2 v. 625 µm2; P &lt; 0.05). Fluorescence images showed higher arbitrary units of fluorescence (auf) for LD in BI. Compared with BT embryos (386 auf v. 280 auf; P &lt; 0.05). These results suggest that BI embryos contain more and smaller LD than BT embryos and the LD described for BI embryo quality grade 1 are larger than those of quality grades 2 and 3, and even though the number of LD in morulae and blastocyst stage embryos are not different LD size is reduced as development occurs. Research funding provided by UNAM-DGAPA-PAPIIT IN200810.

Embryo production and recovery in goats by non-surgical transcervical technique

The objective of this study was to evaluate the efficiency of non-surgical embryo collection in goats. Five lactating Saanen goats were tested alternately in a cross-over design for two treatments (T1 and T2). In both treatments, intravaginal progesterone devices were inserted (day 0/6p.m.) and removed six days later (day 6/18:00p.m.) and 22.5μg cloprostenol were administered by intravulvosubmucosal via (day 0). From day 4 (18:00 p.m.), goats received six decreasing doses of FSH (250–400IU) at 12h interval. Three doses of 50mg flunixin meglumine were administered on days 9, 10, and 11. In T1 and T2, animals received two doses of saline or 250mg of rbST on day 0 and on first detection of estrus, respectively. Goats were mated until the end of estrus and uteri were flushed by cervical route seven days after first mating. The embryo collection interval was 60days. Percentage of flushing medium recovered was close to 100% in both treatments. Total number of ova (18.4±9.4 and 15.7±11.0) and viable embryos (13.4±4.1 and 8.2±3.0) were similar (P>0.05) for T1 and T2, respectively. Short-term progesterone protocol was efficient for superovulation in lactating Saanen goats but association with rbST did not enhance embryo recovery or quality. Non surgical flushing by cervical via was efficient to recover flushing medium and embryos in goats.

228 DOMINANT FOLLICLE ABLATION IMPROVES SUPEROVULATORY RESPONSE AND EMBRYO YIELD IN BUFFALOES SUBJECTED TO INTEGRATED SINGLE-OVULATION EMBRYO TRANSFER - MULTIPLE-OVULATION EMBRYO TRANSFER PROGRAM

Effect of the removal of inhibitory consequences of a dominant follicle (DF) on the recruitment, growth and ovulation of follicles in cyclic buffaloes induced to superovulate, was evaluated. Eight elite, multiparous, donor Murrah buffaloes, 80 to 120 days postpartum, were included in an integrated single-ovulation embryo transfer–multiple-ovulation embryo transfer (SOET-MOET) schedule. Superovulation with FSH (Folltropin® NIH-FSH-P1, Bioniche Animal Health, Belleville, ON, Canada) was preceded by nonsurgical single embryo (SOET) recovery on Day 5 or 6 after prostaglandin F2α (Lutalyse®, Pfizer, Madison, NJ, USA)-induced oestrus and AI (Day 0). For MOET, Folltropin was administered from Day 9 p.m. through to Day 13 p.m. in a twice-daily descending dose schedule (0–5, 5–4, 4–3, 3–2, 2–1 mL; total dose of 580 mg of NIH-FSH-P1). A dose of 25 mg of Lutalyse was administered on Day 12 a.m. and p.m. Donors were inseminated with proven semen on Day 14 a.m. and p.m. and Day 15 a.m. Nonsurgical embryo collection was carried out on Day 19 or 20 followed by administration of Lutalyse on Day 25. Following sexual rest for one complete oestrous cycle, Day 5 or 6 SOET was repeated. On Day 9, ultrasound-guided follicle aspiration was used to ablate the DF and the second MOET treatment was initiated at the same time. Mean diameter of DF at the start of superovulation treatment on Day 9 was similar between the first (12.5 ± 0.66 mm) and second (12.2 ± 0.33 mm) MOET cycles. At AI, mean number (9.6 ± 1.43 vs 13.4 ± 1.87) of follicles &gt;9 mm were higher (P &lt; 0.05) in the second (ablation) MOET cycle, though their mean diameters were similar (11.7 ± 0.21 vs 12.3 ± 0.21 mm; P = 0.17). On the day of ova/embryo collection, almost similar numbers of large follicles (4.1 ± 0.93 and 4.4 ± 0.84), with mean diameters of 12.9 ± 0.74 and 13.4 ± 0.83 mm, were observed for the first and second MOET, respectively. Mean (± SEM) numbers of corpora lutea (3.9 ± 0.81 and 4.9 ± 0.61; P = 0.19) and transferable quality embryos (0.6 ± 0.37 and 1.6 ± 0.37; P = 0.077) were counted in the first and second (ablation) MOET cycles, respectively. These preliminary findings suggest that ablation of DF before start of superovulatory treatment may be beneficial in improving the yield of transferable embryos in a buffalo MOET program. Director, CIRB, is acknowledged for the experimental facilities.

117 SERUM PROGESTERONE CONCENTRATION AND EMBRYO DIAMETER ON DAY 7 POST-OVULATION IN MARES

Monitoring serum progesterone (P4) concentrations has been a routine practice for determining phases of the oestrous cycle in mares. The use of maternal serum P4 concentration to estimate embryo size would be beneficial in determining the proper collection time for equine embryos destined for cryopreservation and transfer. Therefore, the objective of this study was to determine if embryo diameter was correlated with maternal serum P4 concentration on Day 7 post-ovulation. Light horse mares of various breeds (n = 40) were used in this study. The mares were in good body condition and ranged in age from 3 to 18 yrs. Oestrus detection was performed first with a stallion followed by reproductive tract examinations via transrectal ultrasonography to predict ovulation. Blood samples were collected via jugular venipuncture before artificial insemination (AI) (Day 0) and again on Day 7 post-ovulation. Blood samples were allowed to clot, then centrifuged for 10 min at 2600 × g and serum stored at –20°C until assayed. Each donor mare was inseminated with extended fresh semen every other day until ovulation. On Day 7 post-ovulation, a nonsurgical embryo collection was performed on inseminated mares by a single technician using a standard equine uterine flushing procedure. Only blood samples taken from mares in which an embryo was collected were analysed (n = 18). Blood samples were collected from similar noninseminated mares (n = 12) from the same research herd on the day of expected AI (Day 0) and again on Day 7 post-ovulation. Serum progesterone was determined using a P4 fluorometric enzyme immunoassay (FEIA; Tosoh Bioscience, Inc., South San Francisco, CA). Embryo diameter was measured including the zona pellucida using Micron Software (Westover Scientific). Mean P4 concentrations (±s.e.) for all inseminated donors producing an embryo were 0.49 ± 0.1 ng mL–1 at Day 0 and 12.9 ± 1.2 ng mL–1 on Day 7 post-ovulation. Mean P4 concentration of donors on Day 7 post-ovulation with embryo diameters of &lt;400 μm (n = 7) was not different from donors producing embryos &gt;400 μm (n = 10) and were 11.2 ± 1.7 ng mL–1 and 14.1 ± 1.7 ng mL–1, respectively. One mare produced viable twin embryos (284 and 373 μm in diameter) and had a P4 level of 35 ng mL–1 on Day 7 post-ovulation, and was not included in the data analysis. Mean P4 concentrations in the noninseminated mares were 0 0.68 ± 0.2 ng mL–1 on Day 0 and 12.3 ± 1.6 ng mL–1 on Day 7 post-ovulation and were not different from embryo-producing mares regardless of embryo size. From this preliminary study, that there is no evidence that embryo diameter is correlated with maternal progesterone concentrations on Day 7 post-ovulation.

Efficacy of a commercially available post-thaw bovine semen sexing kit in both single-ovulating and hyperstimulated cows

Currently, there are few inexpensive, reliable, effective methods for commercially separating X- and Y-chromosome bearing fresh and frozen bovine sperm. The objective of these experiments was to determine the efficacy of a commercially available post-thaw bovine semen sexing kit, HeiferPlus™ (HP) which claims to alter the sex ratio in favor of female calves following artificial insemination. Three trials included the insemination of hyperstimulated cows with Control or HP-treated semen, non-surgical embryo collection on Day 7, and a combined PCR/dot blot assay to determine embryo sex. Chi-square analysis showed that the Control group produced a greater proportion ( p < 0.0005) of female embryos than the HP group. There were no differences in the proportions of transferable compared with degenerate embryos or in number of ovulations, embryos, and unfertilized ova collected from Control compared with HP groups. When treatments were combined, one of the two bulls used in the hyperstimulation studies produced an overall greater proportion of females ( p < 0.05), suggesting a bull effect. Another trial involved the insemination of cows synchronized via OvSynch ® with fetal sexing via ultrasonography. Results of these studies indicated that HP semen sexing kit did not alter the sex ratio in favor of females in either hyperstimulated or single-ovulating cows; however, potential bull effects may be further evaluated to understand the capacity of HP with semen from specific bulls. Additionally, perhaps the sex of the surviving embryo can be manipulated by the maternal side, through ovarian, hormonal, oviductal, or uterine influences.

260 EFFECTIVENESS OF A COMMERCIALLY AVAILABLE POST-THAW BOVINE SEMEN SEXING KIT IN BOTH MULTIPLE-AND SINGLE-OVULATING COWS

Currently, there is no inexpensive method for commercially separating X- and Y-bearing bovine sperm cells. Because dairy heifer calves are significantly more valuable than bull calves, a reliable method of swaying the bovine sex ratio in favor of females is desirable in the dairy industry. The objective of these experiments was to determine the efficacy of a commercially available post-thaw semen sexing kit, HeiferPlus™ (Emlab Genetics, Arcola, IL, USA), which claims to sway the bovine sex ratio in favor of female calves. Three trials included the insemination of hyperstimulated cows with control or HeiferPlus (HP)-treated semen, nonsurgical embryo collection on Day 7, and a novel combined PCR/Southern blot assay to determine sex. Overall, 271 embryos were collected and a sex was assigned to 265 of them. Chi-square analysis showed that the control group produced a significantly higher proportion (P &lt; 0.005) of female embryos than the HP group (65.0% and 43.0%, respectively). There was no difference in the proportion of transferable versus degenerate embryos, and ANOVA showed no difference in the number of ovulations, embryos, and unfertilized oocytes collected in the control versus the HP groups. Another trial involved the artificial insemination of cows synchronized via OvSynch� (ABS Global, Deforest, WI, USA) followed by fetal sexing via ultrasonography between Days 55 and 58. Of the 152 cows inseminated, 51.3% were pregnant at Day 35. Of the cows inseminated with HP-treated semen, 54.5% became pregnant and 48.0% of the controls were pregnant. HP-treated cows resulted in 54.8% male (23/42) and 45.2% female (19/42) fetuses. Control cows had 52.8% male (19/36) and 47.2% female (17/36) fetuses at 58 days of gestation. Calving records confirmed the ultrasound sexing data and showed that three controls and one HP calf died after 58 days of gestation, and one control and one HP calf died within 2 weeks following birth. Chi-square analysis showed no significant difference in the sex ratio. Semen from six bulls was used in this trial, and pregnancy rates among bulls did not differ (P &gt; 0.05). Computer-assisted semen analysis showed no significant difference in motility or progressive forward motility of control versus HP-treated sperm in any of the bulls. Results of these studies indicated that the HeiferPlus semen sexing kit did not sway the sex ratio in favor of females in either multiple- or single-ovulating cows. In the hyperstimulated cows, the control group actually produced a significantly higher proportion of female embryos than the HP group. No differences in embryo production or embryo grade were observed. Further research is needed to investigate the effects of semen incubation or manipulation on the sex ratio of cattle.

Exogenous eFSH, follicle coasting, and hCG as a novel superovulation regimen in mares

The objective of this study was to evaluate various equine follicle-stimulating hormone (eFSH) treatment protocols and the effect of “follicle coasting” on ovulation and embryo recovery rates in mares. Cycling mares (n = 40) were randomly assigned to one of four groups 7 days after ovulation: (1) 12.5 mg eFSH twice daily until follicles were 35 mm or larger; (2) 12.5 mg eFSH twice daily until follicles were 32 mm or larger; (3) 12.5 mg eFSH twice daily for 3.5 days followed by 12.5 mg eFSH enriched with luteinizing hormone (LH) twice daily until follicles were 35 mm or larger; and (4) 25 mg eFSH once daily until follicles were 32 mm or larger. Mares in groups 1 and 3 were injected with human chorionic gonadotropin (hCG) (2500 IU intravenously) at the end of eFSH treatment, whereas mares in groups 2 and 4 were given hCG approximately 42 and 54 hours, respectively, after the last eFSH treatment (“follicle coasting”). Nonsurgical embryo collection was performed 6.5 to 7.5 days after ovulation. Each mare experienced a nontreated estrous cycle before being reassigned to a second treatment. Ovulation rates for mares in treatment groups 1 to 4 were 3.3 ± 0.4, 4.1 ± 0.4, 3.5 ± 0.4, and 2.8 ± 0.4 (mean ± SEM; P < .05), respectively. One or more embryos were recovered from more than 80% of mares in each treatment group, and embryo recovery rate per flush was similar among treatment groups (1.9 ± 0.3, 2.6 ± 0.3, 1.9 ± 0.3 and 1.9 ± 0.3, respectively; P > .05). The overall embryo recovery rate was 2.1 ± 1.5 embryos per flush. In summary, ovulation rate was higher for mares treated with eFSH (3.4 ± 0.4) compared with non-treated controls (1.1 ± 0.2). Ovulation rate in mares in which hCG was delayed (follicle coasting) was higher ( P < .05) when treatments were given twice per day versus once per day. Administration of equine luteinizing hormone (eLH) in conjunction with eFSH did not have an advantage over mares treated only with eFSH.

215 COMPARISON BETWEEN CONVENTIONAL DIRECT TRANSFER FREEZING AND VITRIFICATION FOR THE CRYOPRESERVATION OF IN VIVO EMBRYOS FROM BRAHMAN CATTLE

There is a need to develop an efficient cryopreservation technique for Brahman cattle embryos that would lead to improved success in the propagation of this breed. The objective of this study was to compare the post-thaw pregnancy rates in recipient cows after nonsurgical transfer of Brahman in vivo-derived embryos frozen in ethylene glycol (EG) and by a new vitrification (VT) method. Prior to the initial in vivo study, hatching rates were recorded 72 h post-thaw on 3 treatment groups (No treatment: 43/55, 78%; EG: 39/83, 47%; and VT: 33/103, 32%) of IVF embryos to determine if development could be achieved. This warranted Phase I: In vivo embryos were produced by multiple ovulation and nonsurgical embryo collections from Brahman cows 7 days post-estrus and AI. Embryos in morula/blastocyst stage of development were graded and maintained in holding medium (Vigro; Bioniche Animal Health, Inc., Belleville, Ontario, Canada) after collection, until randomly allocated for EG or VT protocols. Vitrification was performed using experimental media provided by a commercial company (V1: Vigro + 5 M EG; V2: Vigro + 7 M EG + 0.5 M Galactose + Ficoll; and Diluent: Vigro + 0.5 M Galactose) and by direct plunging into LN2 (Walker et al. 2005 Reprod. Fertil. Dev. 17, 153). Freezing was performed at 0.5�C min-1 from -6�C to -32�C, using a commercially available medium (Vigro Freeze Plus; Bioniche). All embryos were packaged in sterile 0.25-mL plastic straws to allow for direct transfer (DT). Embryos were stored in LN2, and later thawed and nonsurgically transferred to synchronized recipient cows on Day 7 of their cycle. EG straws were air-thawed 5 s and then thawed in 30�C H2O for 10 s. VT straws were air-thawed 10 s and then thawed in 37�C H2O for 20 s prior to shaking them down to mix columns. Pregnancy results evaluated by ultrasound on Day 35 for the presence of a fetus from 4 replicates were 27% for EG (3/11) and 8% for VT (1/13) embryos. Problems with VT straws cracking during air thawing led to a second trial (Phase II) which changed the direct plunging technique for VT to a vapor freeze of 1 to 15 min prior to plunging into LN2. Pregnancy results from 16 transfers were 38% for EG (3/8) and 50% for VT (4/8) embryos, with no straws cracking. Although the number of observations is small, the results obtained showed that VT-DT is satisfactory for producing pregnancies comparable to EG-DT. Further research is required to confirm the results obtained in this preliminary study, and to test whether this method will allow the successful production of pregnancies under different environmental and field conditions.