Yield Of Transferable Embryos Research Articles

Embryo yield and quality in superovulated Simmental donor cows following oral supplementation with different sources of omega-3 and omega-6 fatty acids

Owing to contradictory results with respect to the yield and quality of embryos after supplementation with omega-3 fatty acids, this study sought to determine the effect of an oral supplement containing sources of omega-3 and omega-6 fatty acids on the ovarian response and embryo quality after superovulation. Cows were allocated to receive one of three oral supplement regimens. In the control (CO) group (n = 9), cows were fed a control supplement without fatty acids. The cows in the soybean oil (SO) (n = 8) and linseed oil (LO) (n = 8) groups received supplements containing SO or LO, respectively. Supplements were administered for 28 days, commencing 7 days before the onset of estrus synchronization until the day of embryo collection. The estrous cycle of all cows was synchronized by inserting a controlled internal drug release (CIDR) device (progesterone 1.38 g), followed by the administration of estradiol benzoate 2.5 mg. The CIDR device was withdrawn 7 days after insertion, followed by intramuscular administration of dinoprost 25 mg. Superovulation was induced via the administration of 152 mg of follicle-stimulating hormone (Folltropin-V) in tapered doses, starting 72 h before CIDR withdrawal. The embryos were collected 6 days after artificial insemination. The estrous response, onset of estrus after CIDR withdrawal, and number of ovulations were similar among the experimental groups (p > 0.05). The mean number of transferable embryos (grades 1 and 2) was higher in the LO group compared to the CO group (9.0 vs 3.2; p < 0.05). Although the number of transferable embryos (grades 1 and 2) was greater in the LO group than that in the SO group, the difference was not statistically significant (9.0 and 4.4; p > 0.05). In conclusion, oral supplementation with LO for 28 days increased the yield of transferable embryos, which could potentially improve the profitability of embryo transfer programs in 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.

Embryo production after superovulation of bovine donors with a reduced number of FSH applications and an increased eCG dose

This study evaluated embryo production after superovulation (SO) with a reduced number of FSH applications and increased eCG dose in 26 Bos taurus × Bos indicus donors. On Day 0, donors received an intravaginal device (CIDR) with 1.9 g of progesterone plus 2.5 mg of estradiol benzoate and 50 mg of progesterone via IM. On Day 4, donors were randomly allotted to one of three SO treatments: 1) 455 IU of Folltropin +400 IU of eCG (n = 9), 2) 350 IU of Folltropin +600 IU of eCG (n = 9), and 3) 500 IU of Pluset + 600 IU of eCG (n = 8). In treatment 455 IU of Folltropin +400 IU of eCG, donors received eight IM Folltropin injections in decreasing dose 12 h apart from Day 4 to Day 7. On Day 6, at the same time as the Folltropin, donors received via IM 25 mg of dinoprost tromethamine (PGF2a). On Day 7, the CIDR was removed, and together with the Folltropin, donors received 200 IU of eCG via IM. In treatment 350 IU of Folltropin +600 IU of eCG, donors received four IM Folltropin injections in decreasing dose 12 h apart on Days 4 and 5. On Day 6, donors received via IM 600 IU of eCG in the morning and two doses of 25 mg of PGF2a 12 h apart. On Day 7, the CIDR was removed. Donors from treatment 500 IU of Pluset +600 IU of eCG received four IM Pluset injections in decreasing dose 12 h apart on Days 4 and 5. On Day 6, donors received via IM 600 IU of eCG in the morning and two doses of 25 mg of PGF2a 12 h apart. On Day 7, the CIDR was removed. In the morning of Day 8, donors from the three treatments received 0.25 mg of GnRH via IM. Artificial insemination was performed on Day 8 (pm) and Day 9 (am). Embryos were collected on Day 15. Variables evaluated were number of CL before embryo collection, number of structures recovered, transferable embryos, degenerate embryos and unfertilized oocytes, recovery rate, and viability rate. There was no difference in any variable among treatments (P > 0.05). In conclusion, replacement of four Folltropin or Pluset injections from a conventional eight FSH-injection SO protocol, by a single injection of 600 IU of eCG, is a good alternative to reduce donor handling without decreasing yield of transferable embryos.

Relationship among circulating anti-Müllerian hormone, insulin like growth factor 1, cadmium and superovulatory response in dairy cows

The objectives of this study were 1. to determine the associations among circulating anti-Mullerian hormone (AMH), insulin like growth factor 1 (IGF1) and cadmium (Cd) concentrations of lactating Holstein cows at the time of superovulation and 2. to determine the effect of circulating AMH, IGF1 and Cd concentrations on the superovulatory response in Holstein dairy cows. Holstein cows (n = 30) were milked thrice daily and housed and fed in free stall barn as a separate group. All animals were synchronized for superovulation and flushed. Three blood samples for AMH, IGF1 and Cd analysis were collected prior to superovulation, at estrus and at the time of embryo collection. The concentrations of blood makers prior to superovulation were highly correlated to superovulatory response. Circulating concentrations of AMH, IGF1 prior to superovulation were negatively correlated to Cd concentrations (P < 0.05). There was no correlation between circulating concentrations of AMH and IGF1. The number of corpus luteum (r = 0.71), total embryo (r = 0.67), total transferable embryo (r = 0.51) and total grade 1 embryo (r = 0.5) were positively correlated to AMH concentrations (P < 0.05). There was a trend for negative correlation found between circulating cadmium concentrations and total grade 1 embryo yield (P < 0.1). When cows were classified into quartiles (Q) of circulating AMH concentration, number of corpus luteum, and total embryos, total transferable embryos and total grade 1 embryos yield was significantly different for AMH quartiles. The superovulatory response parameters evaluated were increased with increased AMH concentrations; particularly we observed a >2-fold difference between first and fourth AMH quartiles in total transferable embryo yield and total grade 1 embryo yield. In conclusion, circulating AMH concentration was strongly associated with superovulatory response. Measuring AMH before enrolling cows in superovulation programs will likely allow practitioners to improve numbers of embryos produced and, thereby, reduce costs per embryo produced.

Neurotrophins and their specific receptors in the oviduct of Japanese quail (Coturnix coturnix japonica)

The objectives of this study were 1. to determine the associations among circulating anti-Mullerian hormone (AMH), insulin like growth factor 1 (IGF1) and cadmium (Cd) concentrations of lactating Holstein cows at the time of superovulation and 2. to determine the effect of circulating AMH, IGF1 and Cd concentrations on the superovulatory response in Holstein dairy cows. Holstein cows (n = 30) were milked thrice daily and housed and fed in free stall barn as a separate group. All animals were synchronized for superovulation and flushed. Three blood samples for AMH, IGF1 and Cd analysis were collected prior to superovulation, at estrus and at the time of embryo collection. The concentrations of blood makers prior to superovulation were highly correlated to superovulatory response. Circulating concentrations of AMH, IGF1 prior to superovulation were negatively correlated to Cd concentrations (P < 0.05). There was no correlation between circulating concentrations of AMH and IGF1. The number of corpus luteum (r = 0.71), total embryo (r = 0.67), total transferable embryo (r = 0.51) and total grade 1 embryo (r = 0.5) were positively correlated to AMH concentrations (P < 0.05). There was a trend for negative correlation found between circulating cadmium concentrations and total grade 1 embryo yield (P < 0.1). When cows were classified into quartiles (Q) of circulating AMH concentration, number of corpus luteum, and total embryos, total transferable embryos and total grade 1 embryos yield was significantly different for AMH quartiles. The superovulatory response parameters evaluated were increased with increased AMH concentrations; particularly we observed a >2-fold difference between first and fourth AMH quartiles in total transferable embryo yield and total grade 1 embryo yield. In conclusion, circulating AMH concentration was strongly associated with superovulatory response. Measuring AMH before enrolling cows in superovulation programs will likely allow practitioners to improve numbers of embryos produced and, thereby, reduce costs per embryo produced.

Quality and developmental rate of embryos produced with sex-sorted and conventional semen from superovulated dairy cattle

This study investigated the effect of sex-sorted semen compared with conventional semen on the outcome of embryo recovery, placing special emphasis on the quality, and developmental stage of embryos. Data were analyzed for 443 embryo collections with sex-sorted semen (SEX group) and 1528 with conventional semen (CONV group) in superovulated dairy heifers and cows. The insemination protocol for conventional semen included two inseminations, comprising a total dose of 30 million sperm passing into the uterine body. For sex-sorted semen, two (30%) to three (70%) deep uterine inseminations were performed, the total dose ranging from eight to 12 million sperm. The data were analyzed separately for heifers and cows. The total number of recovered structures was similar among the groups. The number of viable embryos decreased in the SEX groups compared with the CONV (with 1.4 and 3.2 fewer embryos in heifers and cows, correspondingly, P < 0.001), and correspondingly the proportions of unfertilized ova and degenerated embryos increased in the SEX groups (P < 0.001). The proportion of unsuccessful collections, yielding no transferable embryos, increased in the SEX groups for both heifers (from 7.2% to 11.2%, P = 0.025) and cows (from 9.0% to 20.7%, P < 0.001). Regarding the quality of viable embryos, the quality grades were superior in the CONV group compared with the SEX group for heifers (P < 0.001) and cows (P < 0.001). The proportion of grade 1 embryos decreased by 6.5 percentage points in heifers and 11.9 percentage points in cows when sex-sorted semen was used. Correspondingly, the proportions of grade 2 and 3 embryos increased in heifers and cows when sexed semen was used. The mean developmental stages of embryo collections were numerically slightly lower in the SEX group. In heifers, the delay in developmental stage was statistically significant (P = 0.001), but in cows, there was only a tendency toward that (P = 0.067). In conclusion, sex-sorted sperm decreased the transferable embryo yield and increased the risk of a recovery yielding no transferable embryos. Furthermore, use of sex-sorted semen decreased the proportion of grade 1 embryos. In addition, it also seemed to delay embryonic development, although the delay in embryonic development was minimal and its biological relevance remains undefined. Despite the compromised embryo production, taken into account the optimization of recipient resources, the use of sex-sorted semen is advantageous, especially in superovulated heifers, which are of most importance in the modern breeding strategies using genomic selection.

108 COMPARATIVE EFFICIENCY OF GONADOTROPIN-RELEASING HORMONE AND LUTEINIZING HORMONE IN THE INDUCTION OF OVULATION IN SUPEROVULATED ALPACAS

Alpacas are induced ovulators, responding to copulation and (or) exogenous application of ovulation-inducing hormones. The objective of this study was to determine the efficiency of the injection of a gonadotropin-releasing hormone (GnRH) agonist versus LH in the induction of ovulation and the presence and size of non-ovulated follicles at the time of embryo collection and its relationship to the yield of transferable embryos in superovulated alpacas. Twenty-one adult (3 to 7 years old) female alpacas under extensive grazing at 4300 m elevation in the Peruvian Andes that had been synchronized and treated for superovulation were induced to ovulate 6 days after the application of the superovulatory hormone (1000 IU of eCG, Folligon®, Intervet International BV, Boxmeer, the Netherlands) by mating with fertile males and injection immediately after copulation of either an IM dose of 0.0084 mg of buserelin acetate (Buserelina Zoovet®, Laboratorio Zoovet, Santa Fe, Argentina) to 10 alpacas (T1) or an IM dose of 5-mg Armour standard of LH (Lutropin®, Bioniche Animal Health, Belleville, ON, Canada) to 11 alpacas (T2). All alpacas had a second mating 12 h after the first mating and were subjected to ovarian inspection by ultrasonography and previous embryo collection by nonsurgical transcervical embryo flushing 6.5 days after the first mating. On average, the embryo recovery rate for T1 was 34.6% and there were 7.8 ± 3.99 corpora lutea (CL), 2.7 ± 4.08 collected embryos, and 3.6 ± 2.95 total, 0.5 ± 0.85 small (&lt;6 mm), 1.8 ± 1.99 medium (≥6 but ≤14 mm), and 1.3 ± 2.11 large (≥15 mm) non-ovulated follicles. For T2, the embryo recovery rate was 59.4% and there were 6.73 ± 1.49 CL, 4.0 ± 2.57 collected embryos, and 0.64 ± 0.81 total, 0.0 ± 0.0 small, 0.36 ± 0.67 medium, and 0.27 ± 0.47 large non-ovulated follicles. The differences between treatments were nonsignificant (P &gt; 0.05) for all the parameters; however, there was a clear tendency for a better recovery rate, more embryos collected, and lower number of non-ovulated follicles in T2. The Pearson correlation coefficient between the presence of large follicles in the ovaries at the time of embryo collection and the total number of embryos collected was negative (r = –0.26) and highly significant (P ≤ 0.001). The use of LH for ovulation induction of superovulated alpacas seems to be more advisable than the use of GnRH agonist; further comparisons with larger number of observations per treatment are recommended. This study was financed by the Peruvian Fund for Innovation, Science and Technology (FINCYT).

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.

In vitro developmental competence of buffalo oocytes collected at various stages of the estrous cycle

The objective was to determine the in vitro developmental competence of buffalo oocytes collected from abattoir-derived ovaries at various stages of the estrous cycle and follicular status. In Experiment 1, ovaries ( n = 476 pairs) were collected and divided into the following five groups: (a) ovaries with a corpus hemorragicum and no dominant follicle (CH-NO-DF); (b) ovaries with a mature functional corpus luteum (CL) and a dominant follicle (CL-DF); (c) ovaries with a mature functional CL and no dominant follicle (CL-NO-DF); (d) ovaries with a regressing CL and a dominant follicle (RCL-DF); and (e) ovaries without any luteal structures and only small follicles (ANEST). In Experiment 2, 144 pairs of ovaries with a CL (or regressing CL) and a dominant follicle were collected and follicles were classified as dominant, largest subordinate, and subordinate. In both experiments, the dominant follicle was defined as any follicle >10 mm in diameter that exceeded the diameter of all other (subordinate) follicles. Although oocytes were collected from each group of ovaries, only Grades A or B oocytes were used for in vitro embryo production. Cleavage rates were higher ( P < 0.05) from oocytes collected from ovaries in the CH-NO-DF (59.6%) and CL-NO-DF (59.2%) groups than those collected from CL-DF (52.2%) and ANEST (43.6%) groups. The yield of transferable embryos was higher ( P < 0.05) from oocytes collected from CH-NO-DF (27.4%) and CL-NO-DF (24.0%) ovaries than from CL-DF (16.2%), RCL-DF (15.4%), and lowest ( P < 0.05) from ANEST (8.8%). In Experiment 2, oocytes from the dominant follicle had a higher ( P < 0.05) cleavage rate (65.2 %) and transferable embryo yield (30.2%) than those collected from the largest subordinate and subordinate follicles. In conclusion, oocyte competence depended on the morphofunctional state of ovaries. Oocyte development was maximal in pairs of ovaries with a corpus hemorragicum or CL and no dominant follicle; in paired ovaries with a CL and a dominant follicle, development was maximal in oocytes derived from the dominant follicle.

Advanced reproductive technology in the water buffalo

Embryo transfer techniques in water buffalo were derived from those in cattle. However, the success rate is much lower in buffaloes, due to their inherent lower fertility and poor superovulatory response. The buffalo ovary has a smaller population of recruitable follicles at any given time than the ovary of the cow (89% fewer at birth). In addition, estrus detection is problematic. Progress in the field of embryo transfer in water buffalo has been slow, and is primarily due to a poor response to superovulation. The average yield of transferable embryos is less than one per superovulated donor. In vitro embryo production could considerably improve the efficacy and logistics of embryo production. The technique of Ovum Pick Up is superior to superovulation; it can yield more transferable embryos per donor on a monthly basis (2.0 versus 0.6). The feasibility of intergeneric embryo transfer between buffalo and cattle has been investigated. No pregnancy resulted after transfer of 13 buffalo embryos to synchronized Holstein heifers. Preliminary successes with nucleus transfer of Bubalus bubalis fetal and adult somatic nuclei into enucleated bovine oocytes and subsequent development to the blastocyst stage have been reported.

Enrichment of in vitro maturation medium for buffalo ( Bubalus bubalis) oocytes with thiol compounds: Effects of cystine on glutathione synthesis and embryo development

The purpose of this study was to evaluate whether enriching the oocyte in vitro maturation medium with cystine, in the presence of cysteamine, would improve the in vitro embryo production efficiency in buffalo by further increasing the GSH reservoir created by the oocyte during maturation. Cumulus–oocytes complexes were matured in vitro in TCM 199 + 10% FCS, 0.5 μg/ml FSH, 5 μg/ml LH and 1 μg/ml 17β-estradiol in the absence or presence of cysteamine (50 μM), with or without 0.3 mM cystine. In Experiment 1, glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in representative samples of oocytes matured in the four different experimental conditions. In Experiment 2, oocytes were fixed and stained to assess nuclear maturation and normal pronuclear development following IVM and IVF respectively. In Experiment 3, mature oocytes were in vitro fertilized and cultured to assess development to blastocysts. In all supplemented groups the intracytoplasmic GSH concentration was significantly higher than the control, with the highest GSH levels in oocytes matured in the presence of both thiol compounds (3.6, 4.7, 5.4 and 6.9 picomol/oocyte in the control, cysteamine, cystine and cystine + cysteamine groups, respectively; P < 0.05). Cystine supplementation of IVM medium, both in the presence or absence of cysteamine, significantly increased the proportion of oocytes showing two normal synchronous pronuclei following fertilization. In all supplemented groups, cleavage rate was significantly improved compared to the control (55, 66.1, 73.5 and 78.4% in the control, cysteamine, cystine and cystine + cysteamine groups, respectively; P < 0.05). Similarly, blastocyst yield was also increased in the three enriched groups compared to the control (17.1, 23.8, 29.3, 30.9% in the control, cysteamine, cystine and cystine + cysteamine groups, respectively; P < 0.05). Overall, the addition of cystine to a cysteamine-enriched medium resulted in a significant increase of cleavage rate and transferable embryo yield compared to the medium supplemented with only cysteamine.

Prepubertal propagation of transgenic cloned goats by laparoscopic ovum pick-up and in vitro embryo production.

The use of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production was evaluated in the early propagation of cloned goats. Ten kinder goats produced by somatic cell nuclear transfer technology were used as oocyte donors. Half of the donor animals were subjected to LOPU at 2-3 months of age (prior to induction of lactation), whereas the other five goats were subjected to LOPU at 6-7 months of age (following induction to lactation). They were stimulated with 80 mg NIH-FSH-P1 (Folltropin, Vetrepharm, Canada) together with 300 IU eCG (Novormon, Vetrepharm, Canada) administered intramuscularly 36 h prior to LOPU. The number of follicles aspirated and oocytes recovered was higher in the younger group of donors (57 +/- 7 and 41 +/- 4 vs. 28 +/- 2 and 25.8 +/- 2, p < 0.05), however, oocytes from animals in the late prepubertal age showed higher developmental capacity resulting in higher transferable embryo yield (81.4% vs. 67.8%, p < 0.01), pregnancy rate (80% vs. 40%, p < 0.05) and total kids born (27 vs. 15, p < 0.01). In conclusion, LOPU in combination with in vitro embryo production techniques is an efficient method for the early propagation of valuable goats produced by somatic cell nuclear transfer.

Effect of dietary regimens on ovarian response and embryo production of sheep in tropics

Bharat Merino ewes ( n=24) maintained in a tropical environment were allocated to three dietary treatment groups. The ewes were grazed on pasture either without supplementation (Group 1) and with supplementation of concentrate feed at the rate of 150 g per ewe per day (Group 2) or 300 g per ewe per day (Group 3), respectively. Estrus was synchronized with two injections of prostaglandin (PGF 2α) 10 mg at 10 days interval. Superovulation treatment commenced 3 days prior to the second injection of PGF 2α. Each ewe received a total dose of 5.4 mg FSH every 12 h over 4 days and PMSG (200 IU) once at the start of the treatment. The ewes in estrus were exposed to fertile rams. Ovarian examination and recovery of embryos and ova were based on laparoscopy and laparotomy between days 3 and 6 after mating. Data for onset of estrus, duration of estrus, number of corpora lutea (CL), number of unovulated large follicles (LF), embryo recovery rate, embryo quality and fertilization were recorded. Onset of estrus after the second dose of PGF 2α and duration of estrus did not differ significantly among the three groups. Number of ovulations, ovarian response (CL+LF), embryo recovery and yield of transferable embryos for all the dietary treatment groups were similar ( P>0.05). It is concluded that the dietary flushing of Bharat Merino ewes prior to superovulation and mating did not influence the estrus response, superovulatory response, embryo recovery or yield of transferable embryos.

Effect of mating system, flushing procedure, progesterone dose and donor ewe age on the yield and quality of embryos within a MOET program in sheep

Multiple ovulation and embryo transfer (MOET) has the potential to increase the rates of genetic improvement in sheep. However, better realization of this potential requires a higher yield of transferable embryos. Thus we investigated some factors that may contribute to high embryo yield and quality under field conditions, as part of an ongoing MOET program. Comparison of the effects of 2 breeding systems (natural service plus laparoscopic intrauterine AI vs natural service only) on embryo yield and quality indicated that while AI did not affect embryo recovery, it significantly (P<0.05) improved fertilization rate and embryo quality, and increased (P<0.05) embryo survival rate after transfer to recipients. Two flushing procedures (the original semi-laparoscopic and a modified version) were compared for effects on embryo recovery. The modifications made to the original collection method increased (P<0.001) embryo recovery from 69.0 ± 2.4 to 83.2 ± 0.6%. The effects of the progestagen priming dosage during superovulatory treatment and ewe age on MOET outcome were also investigated. Donor ewes primed with 30-mg progestagen sponges came into estrus 1.9 h earlier (P<0.05) than those primed with 45-mg sponges, but there was no difference in ovulation rate or embryo recovery, or in embryo survival after transfer between the 2 regimens. However, Chi-square analysis indicated a significant benefit in favor of the higher progesterone dose on both fertilization (P<0.01) and embryo quality (P<0.001). Age of donor ewe did not significantly affect the timing of estrus, fertilization rate or embryo survival after transfer. While adult ewes had higher (P<0.05) ovulation rates and embryo yields, shearling ewes produced a much higher proportion of Grade 1 embryos (P<0.05).

Active immunization with a synthetic fragment of pig inhibin alpha-subunit increases ovulation rate and embryo production in superovulated ewes but season affects its efficiency.

Two experiments were designed to determine the effects of active immunization against one of two synthetic peptides from humans (inhibin-like peptide) or pigs (inhibin alpha-subunit) on antibody titres, ovulation rate and embryo production in ewes superovulated with 16 U ovine FSH. In Expt 1, during the breeding season, 30 ewes were subdivided into three groups: group I served as the non-immunized control; group II was immunized against inhibin-like peptide (100 micrograms inhibin-like peptide equivalent, followed by three booster injections); group III was immunized against pig inhibin alpha-subunit conjugated to human serum albumin (96 micrograms for the primary administration and 46 micrograms for the booster). In Expt 2, the efficiency of immunization against pig inhibin alpha-subunit on ovarian response and embryo production was evaluated during the non-breeding season in two groups of ewes (n = 12): group IV was a non-immunized control; Group V was immunized against pig inhibin alpha-subunit. During the breeding season, the ewes immunized against pig inhibin alpha-subunit showed higher antibody titres compared with the group immunized against inhibin-like peptide (P < 0.01) and a significant increase in ovulation rate (12.1) compared with both the control (5.0; P < 0.05) and the inhibin-like peptide-immunized group (3.1; P < 0.01). Immunization against pig inhibin alpha-subunit increased transferable embryo yield 4.5-fold (6.7 versus 1.5; P < 0.01) and improved embryo quality (94.6 versus 40.6%; P < 0.01). During the non-breeding season, immunization against pig inhibin alpha-subunit enhanced ovulation rate from 2.6 in the controls to 9.4 (P < 0.01) but did not affect transferable embryo production (3.9 versus 2.1; P > 0.05) and significantly lowered their quality (54.1 versus 100%; P < 0.01). In conclusion, active immunization against pig inhibin alpha-subunit can improve superovulatory response during the breeding season, while it appears to be unable to increase embryo yield during the seasonal anoestrus.

Superovulation and embryo production in ewes using a commercial p-FSH

The effectiveness of a commercial porcine pituitary extract (p-FSH) with a 1:1 FSH/LH ratio was assessed on ovarian response and embryo yield in ewes. During the breeding season, 60 Altamurana ewes were synchronized with FGA-impregnated intravaginal sponges and subdivided into four treatment groups ( n = 15). Beginning 24 h before sponge removal, ewes were treated with 250 (Group 1), 500 (Group 2), 750 (Group 3) or 1000 IU p-FSH (Group 4) administered in a series of four decreasing doses over a period of 36 h. The ewes were monitored for estrus every 4 h, with ovarian response and embryos evaluated on the seventh day following sponge removal. No differences were found among the treatments in incidence of estrus and ovulation. Although treatment with 250 IU p-FSH produced the fewest ovulations, animals in this group yielded a higher ( P < 0.01) number of viable embryos, a lower ( P < 0.01) number of unfertilized ova, and a lower ( P < 0.05) incidence of unovulated follicles and abnormal corpora lutea when compared with animals treated with 750 or 1000 IU p-FSH, while treatment with 500 IU p-FSH yielded intermediate results. It is concluded that the commercial porcine pituitary extract (Pluset) at moderate doses (250 IU) is suitable for superovulation in ewes as it induces a high ovulatory response and transferable embryo yield.

Follicle aspiration prior to superovulation in cattle: A field study

Embryo transfer techniques in water buffalo were derived from those in cattle. However, the success rate is much lower in buffaloes, due to their inherent lower fertility and poor superovulatory response. The buffalo ovary has a smaller population of recruitable follicles at any given time than the ovary of the cow (89% fewer at birth). In addition, estrus detection is problematic. Progress in the field of embryo transfer in water buffalo has been slow, and is primarily due to a poor response to superovulation. The average yield of transferable embryos is less than one per superovulated donor. In vitro embryo production could considerably improve the efficacy and logistics of embryo production. The technique of Ovum Pick Up is superior to superovulation; it can yield more transferable embryos per donor on a monthly basis (2.0 versus 0.6). The feasibility of intergeneric embryo transfer between buffalo and cattle has been investigated. No pregnancy resulted after transfer of 13 buffalo embryos to synchronized Holstein heifers. Preliminary successes with nucleus transfer of Bubalus bubalis fetal and adult somatic nuclei into enucleated bovine oocytes and subsequent development to the blastocyst stage have been reported.

The development of a monoclonal antibody against PMSG for veterinary application

The development of Neutra-PMSG, a monoclonal antibody (mAb) against PMSG for commercial veterinary application, is discussed. The mAb is of murine origin and is produced in a hollow fibre dialysis system. The product is indicated for the neutralisation of residual PMSG in superovulated donor cows, thus increasing the yield of transferable embryos. From the range of potential candidates one mAb was selected by means of in vivo and in vitro neutralisation tests. The mAb was further characterised by determining its effect on the FSH and LH bioactivity of PMSG. It was shown that the mAb is highly specific and it does not cross react with other gonadotrophic hormones of bovine or human origin. Some aspects of the pharmacology, the production and the quality control of Neutra-PMSG are reported.

The effect of type and number of fsh injections on superovulation in ewes

One of the major limiting factors in practical exploitation of embryo transfer is the lack of a predictable and reliable supply of embryos on a regular basis. Superovulation requires the stimulation of significantly increased numbers of pre-ovulatory follicles by the administration of gonadotrophins mimicking the effect of follicle stimulating hormone (FSH). Ewes can be effectively superovulated using gonadotrophins given towards the end of a 12 to 14-day progestagen treatment, initiated without reference to the stage of the oestrous cycle (Boland et al., 1983). However, as in cattle (Goulding et al., 1991) there is a large variation in the follicular and ovulatory response in sheep. Although crude horse pituitary extracts will induce superovulation effectively in sheep, the lack of availability means that they are rarely used. The LH content in commercial FSH preparations has been indicated as contributing to some of the variation in the yield of transferable embryos in cattle following superovulation (Murphy et al., 1984). Recent preliminary data from this laboratory indicates that pFSH with a high LH:FSH ratio will induce superovulation in cattle and sheep. However, pFSH is normally given in multiple injections over 3 to 4 days which makes administration difficult. Therefore, an experiment was designed to assess the efficacy of a pFSH preparation with a high LH:FSH ratio to induce superovulation in sheep when given in either 4 or 6 injections. A second experiment compared the use of an ovine and porcine FSH preparation during superovulation in ewes.

The effect of type and number of fsh injections on superovulation in ewes

One of the major limiting factors in practical exploitation of embryo transfer is the lack of a predictable and reliable supply of embryos on a regular basis. Superovulation requires the stimulation of significantly increased numbers of pre-ovulatory follicles by the administration of gonadotrophins mimicking the effect of follicle stimulating hormone (FSH). Ewes can be effectively superovulated using gonadotrophins given towards the end of a 12 to 14-day progestagen treatment, initiated without reference to the stage of the oestrous cycle (Boland et al., 1983). However, as in cattle (Goulding et al., 1991) there is a large variation in the follicular and ovulatory response in sheep. Although crude horse pituitary extracts will induce superovulation effectively in sheep, the lack of availability means that they are rarely used. The LH content in commercial FSH preparations has been indicated as contributing to some of the variation in the yield of transferable embryos in cattle following superovulation (Murphy et al., 1984). Recent preliminary data from this laboratory indicates that pFSH with a high LH:FSH ratio will induce superovulation in cattle and sheep. However, pFSH is normally given in multiple injections over 3 to 4 days which makes administration difficult. Therefore, an experiment was designed to assess the efficacy of a pFSH preparation with a high LH:FSH ratio to induce superovulation in sheep when given in either 4 or 6 injections. A second experiment compared the use of an ovine and porcine FSH preparation during superovulation in ewes.