CIDR Withdrawal Research Articles

Monitoring changes in back fat thickness and its effect on the restoration of ovarian activity and fertility in Bos indicus cows.

With the objective of testing the hypothesis if animals with a stable layer of body fat (FAT) during the peripartum have a better chance of becoming pregnant after calving, fifty-nine multiparous Brahman cows in their last trimester of pregnancy were used. Animals averaged four parturitions and were stocked at a rate of 1.25 animal units per hectare and divided into two groups depending on the time postpartum (dpp) that the intravaginal releasing device CIDR was inserted; Group 1 (<30dpp; n=30) received the implant at 25.2±4.21 and withdrawn 9days later. Group 2 (≥30dpp; n=29) received the CIDR at 38.41±5.8. Animals were AI at detected oestrus until 170dpp and calculated as pregnant at first service or requiring more than one service (1s and >1s), not pregnant but cycling (not pregnant) and those not cycling at all (anestrus). The FAT measurements were taken twice each month from the last trimester of gestation until 96dpp. The onset of ovarian activity was monitored through blood levels of progesterone (P4) at days 14 and 9 prior to CIDR insertion and days 10, 13, 30 and 33 after CIDR withdrawal. Animals pregnant did not have any major changes in their fat thickness. In contrast, cows pregnant in the group ≥30dpp had changes in their FAT homoeostasis, and pregnant animals in the 1s and >1s groups did not show differences in dorsal back fat in the last trimester of pregnancy and early postpartum. In contrast, animals not pregnant and in anestrus FAT values decreased considerably after parturition. Overall, fertility was 49%, but 18% of all the animals remained anestrus losing FAT. Thus, animals with adequate metabolic conditions will have a better chance of pregnancy regardless of the time postpartum when the reproductive programme starts.

164 The Origin of Oocyte, In Vitro-Matured Oocyte With/Without Super-Stimulation, and In Vivo-Matured Oocyte Influence the Timing of Cleavage in Early Embryo and Oxygen Consumption of Blastocyst After IVF in Japanese Black Cow

It has been reported that in vitro- and in vivo-matured oocyte obtained from fully growth follicles have high developmental competence. Furthermore, the timing of cleavage in early embryo after IVF affect pregnancy success after embryo transfer. It is still unknown whether origin of oocyte affects the timing of cleavage. In this study, we examined the influence of oocyte origin on cleavage timing of early embryo after IVF. Japanese Black cows were used as donors. Oocytes derived from non-stimulation follicles (control: CON), fully grown follicles after super-stimulation treatment (SST) and follicles just before ovulation after ovulation-induction treatment (in vivo-matured oocyte: VIVO) were obtained by ovum pick-up (OPU). In the CON group, OPU was conducted on arbitrary days except oestrus. In SST group, dominant follicles were aspirated and a CIDR was inserted into the vagina on Day 0, and then FSH was injected twice a day from the evening of Day 1 to the morning of Day 5 with decreasing doses in total 20 AU. In the evening of Day 4, prostaglandin F2α (0.5 mg of cloprostenol) was administered. On Day 6, SST oocytes were collected after CIDR withdrawl. In the VIVO group, the treatment was carried out as SST until prostaglandin F2α administration, and then CIDR withdrawal and administration of gonadotropin-releasing hormone (GnRH, 0.2 mg of fertirelin acetate) performed on the evening of Day 4 and morning of Day 5, respectively. The VIVO oocytes were collected at 25 to 26 h after GnRH. The CON and SST oocytes were inseminated after 20 to 22 h of IVM, and VIVO oocytes were inseminated at 30 h after GnRH, with 3 × 106 sperm mL−1, respectively. After 6 h of IVF, presumptive zygotes were individually cultured for 168 h, using a well-of-the-well dish (Dai-Nippon-Print, Japan) and were observed by time-lapse cinematography (CCM-4MZS; Astec, Japan) to analyse the cleavage timing of embryos. Oxygen consumption (O2) was measured in blastocysts on 168 hpi with a scaning electrochemical microscopy system (HV-405SP; Hokuto Denko, Japan). Statistical analysis was carried out by Steel-Dwass test for the timing of cleavage and Tukey-Kramer test for O2. In CON (n = 15), SST (n = 25), and VIVO (n = 36), the time of first cleavage was 27.5, 29.1, and 26.1 hpi, that of second cleavage was 38.9, 40.3, and 36.0 hpi, and that of third cleavage was 48.5, 46.1, and 45.9 hpi, respectively. These cleavage times were shorter in VIVO than in CON and SST (P &lt; 0.01). The time interval between first and second cleavage (2nd cell cycle) was shorter in VIVO (10.1; P &lt; 0.01) than CON (11.4) and SST (11.2). The time interval between second and third (3rd cell cycle) were shorter (P &lt; 0.01) in SST (9.4) than in VIVO (10.1), and in VIVO than in CON (10.2), respectively. Consumption of O2 was lower (P &lt; 0.01) in CON (0.61 × 10−14 mol s−1) than in SST (0.94 × 10−14 mol s−1) and VIVO (0.94 × 10−14 mol s−1). These results suggest that the origin of oocyte influences the length of cell cycle and O2 consumption of blastocyst producted in vitro.

Resumption of ovarian function, the metabolic profile and body condition in Brahman cows (Bos indicus) is not affected by the combination of calf separation and progestogen treatment

To evaluate the effect of different calf separation procedures after a progestogen treatment on the resumption of ovarian function, body condition and metabolic profile, 59 multiparous Brahman cows grazing on a mixed grass pasture were studied. No supplementation was given at any time. Body condition score (BCS), fat thickness (FAT) and blood metabolites were measured fortnightly from the beginning of the last trimester of gestation until 96days postpartum. At 30days postpartum all animals received a progesterone (P4)-releasing device (CIDR) which was withdrawn 9days later when prostaglandin F2α was applied. At this time, treatments TW (n=28), where calves were separated from their dams for 48h; RS (n=21), calves were allowed to suckle once a day for 1h; and continuous suckling (CS; n=10). Ovarian function was assessed by blood concentrations of progesterone on days −14, −9, 10, 13, 30 and 33 after CIDR removal. At the end of the experimental period, an average of 20% of the cows had not initiated estrous cycles. There were no changes of FAT or BCS during the last trimester of pregnancy in all cows (P>0.05). During the postpartum period cows of all groups lost (P<0.05) BCS and FAT with a nadir at 60–80days postpartum, regardless of treatment. At 10days after CIDR withdrawal the percentage of cows having ovulations was 75, 61 and 80 (P>0.05) for TW, RS and CS groups. Blood metabolites follow a similar pattern in the three groups. With the conditions of the present study, the method of calf separation after a progestogen treatment, does not affect the resumption of ovarian function or metabolic profile.

Comparison of 2 protocols to increase circulating progesterone concentration before timed artificial insemination in lactating dairy cows with or without elevated body temperature

Two treatments designed to increase circulating progesterone concentration (P4) during preovulatory follicle development were compared. One treatment used 2 intravaginal P4 implants (controlled internal drug-releasing inserts; CIDR) and the other used a GnRH treatment at beginning of the protocol. Lactating Holstein cows that had been diagnosed as nonpregnant were randomly assigned to receive timed artificial insemination (TAI) following 1 of 2 treatments (n = 1,638 breedings): (1) GnRH: CIDR+ 2 mg of estradiol (E2) benzoate + 100 µg of GnRH on d -11, PGF2α on d -4, CIDR withdrawal + 1.0 mg of E2-cypionate + PGF2α) on d -2, and TAI on d 0; or (2) 2CIDR: 2 CIDR + 2 mg of E2-benzoate on d -11, 1 CIDR withdrawn + PGF2α on d -4, second CIDR withdrawn + 1.0 mg of E2-cypionate + PGF2α on d -2, and TAI on d 0. Milk yield was measured daily between d 0 and d 7. Rectal temperature was measured using a digital thermometer at d 0 and 7, and elevated body temperature was defined as an average rectal temperature ≥39.1°C. Pregnancy diagnoses were performed on d 32 and 60 after TAI. We detected no effect of treatments on pregnancy per AI or pregnancy loss regardless of elevated body temperature, body condition score, parity, milk yield, or presence or absence of a corpus luteum (CL) on d -11 or d -4. Pregnancy per AI at 60 d was reduced [elevated body temperature = 22.8% (162/709), no elevated body temperature 34.1% (279/817)] and pregnancy loss tended to increase [elevated body temperature = 20.2% (41/203), no elevated body temperature 14.4% (47/326)] in cows with elevated body temperature. Various physiological measurements associated with greater fertility were also reduced in cows with elevated body temperature, such as percentage of cows with a CL at PGF2α (decreased 7.9%), ovulatory follicle diameter (decreased 0.51 mm), expression of estrus (decreased 5.1%), and ovulation near TAI (decreased 2.8%) compared with cows without elevated body temperature. A greater proportion of cows (30.2%) had a CL at PGF2α in the GnRH treatment [74.1% (570/763)] than in the 2CIDR treatment [56.9% (434/763)]; however, circulating P4 concentration was greater at the time of PGF2α treatment (d -4) for cows 2CIDR (4.26 ± 0.13 ng/mL) than in cows in GnRH (3.99 ± 0.14 ng/mL). Thus, these 2 protocols yield similar fertility results that might be due to somewhat different physiological alterations. Treatment with GnRH increased the proportion of cows with a CL at PGF2α; however, the 2CIDR protocol increased circulating P4 under all circumstances.

470 Effects of eCG administration 4 versus 2 days prior to timed AI on Nellore cows

The objective of this study was to evaluate growth of the dominant follicle (DF) in multiparous Nellore cows treated with equine chorionic gonadotropin (eCG) either 4 or 2 d prior to timed AI (TAI). We hypothesized that early eCG administration improves follicular growth and increase DF diameter at TAI. Cows (n = 137) had their estrous cycle synchronized with an intravaginal device containing 1.9 g of progesterone (CIDR) and 2.0 mg of estradiol benzoate (d 0), 12.5 mg of PGF2α (d 7), CIDR withdrawal and 0.3 mg of estradiol cypionate (d 9), and TAI 48 h later (d 11). On d 7, ovaries were evaluated by ultrasonography and cows were blocked based on DF diameter (< 9 mm vs. ≥ 9 mm). Within block, cows were randomly assigned to receive 300 IU of eCG either on d 7 (eCGD7; n = 64) or on d 9 (eCGD9; n = 73). Diameter of the DF was evaluated by transrectal ultrasonography on d 7, 9, and 11. Follicular growth from d 7 to 9, 7 to 11, and 9 to 11 was analyzed by ANOVA using the GLIMMIX procedure of SAS. Follicular diameter was analyzed by ANOVA for repeated measures and cow was considered a random effect. Follicular growth from d 7 to 11 tended to be greater (P = 0.08) for eCGD7 compared with eCGD9 (4.9 ± 0.2 vs. 4.4 ± 0.2 mm). The overall effect of treatment was explained mostly by DF growth from d 7 to 9 (eCGD7 = 2.6 ± 0.2 vs. eCGD9 = 2.0 ± 0.2 mm; P < 0.01) as growth from d 9 to 11 did not differ (P = 0.44) between treatments (eCGD7 = 2.3 ± 0.2 vs. eCGD9 = 2.4 ± 0.1 mm). Follicular diameter was affected (P < 0.01) by the interaction between treatment and day. Whereas no difference was observed on d 7 (eCGD7 = 9.7 ± 0.2 vs. eCGD9 = 9.6 ± 0.2 mm), DF in eCGD7 cows was larger (P < 0.01) on d 9 (12.3 ± 0.2 vs. 11.5 ± 0.2 mm). In addition, DF on d 11 did not differ (P = 0.12) between treatments (eCGD7 = 14.5 ± 0.3 vs. eCGD9 = 14.0. ± 0.2 mm). Treatment with 300 IU of eCG 4 d prior to TAI improved follicular growth but did not increase overall mean of DF diameter at TAI compared with eCG administration 2 d before TAI in Nellore cows.

200 THE USE OF LONG-ACTING FSH-MAP5 IN SHEEP SUPEROVULATION PROGRAMS

The administration of sustained-release FSH-MAP5 in a 2-injection protocol has been shown to be as effective as a multiple-FSH injection protocol in inducing superovulation in cattle (Bo and Mapletoft 2014 Theriogenology 81, 38) and in sheep (Fry et al. 2016 Reprod. Fertil. Dev. 28, 250); however, the effect on embryo quality in the latter experiment was unclear. The following experiment further investigated the effect of FSH-MAP5 on ovulation rate and embryo quality in a sheep multiple-ovulation embryo transfer (MOET) program conducted in the breeding season. Two hundred sixteen Dohne merino ewes received a 12-day CIDR-S device containing 0.33 g of progesterone (Zoetis, Florham Park, NJ, USA) plus 200 mg of FSH IM (Folltropin-V; Vetoquinol, Belleville, Canada) and 400 IU of eCG IM (Pregnecol: Vetoquinol) in 4 treatment groups. Group 1 (n = 51) received 7 injections (a.m., p.m.) of FSH in saline (2.5, 2.0, 1.5, 1.5, 1.0, 1.0, and 0.5 mL) starting 2.5 days before CIDR withdrawal and 400 IU of eCG in saline at the time of the first FSH injection. Group 2 (n = 53) received 6 injections (a.m., p.m.) of FSH in saline (3.0, 2.0, 1.5, 1.5, 1.0, and 1.0 mL) starting 2.5 days before CIDR withdrawal and 400 IU of eCG in saline at CIDR withdrawal. Group 3 (n = 56) received 3.3 mL of FSH in hyaluronan (50 mg of MAP5, Vetoquinol) and 400 IU of eCG in saline 2.5 days before CIDR withdrawal and 1.7 mL of FSH-MAP5 at 0.5 days before CIDR withdrawal. Group 4 (n = 56) received 3.3 mL of FSH-MAP5 at 2.5 days before CIDR withdrawal, 1.7 mL of FSH-MAP5 at 0.5 days before CIDR withdrawal, and 400 IU of eCG in saline at CIDR withdrawal. Ewes were inseminated with semen collected and pooled from 5 rams at 36 to 40 h after CIDR withdrawal. Donor ewes were slaughtered 6 days after AI, corpora lutea were counted on the ovary, and ova/embryos were collected. Data for corpora lutea, total ova/embryo, and transferable embryo was analysed by the Kruskal–Wallis test and differences between groups were determined by the Dunn’s test. Results are shown in Table 1. There were no differences in the mean number of ovulations or total ova/embryo collected between any group (P &gt; 0.05); however, those receiving eCG at the time of CIDR withdrawal (Groups 2 and 4) had significantly more transferable embryos than those receiving eCG at the time of the first FSH injection (Groups 1 and 3). In conclusion, the administration of the sustained-release FSH-MAP5 in a 2-injection protocol in sheep was as effective as a multiple-FSH injection protocol in inducing multiple ovulations and yielded similar number of transferable embryos, whereas the administration of eCG at the time of CIDR withdrawal resulted in more transferable embryos, possibly by improving fertilisation rates. Table 1. Results1

Applicability of Day 0 superovulation protocol in Boer goats

This study evaluated the applicability of the Day 0 superovulation protocol in Boer goats by comparing it to a traditional pFSH protocol. Twenty Boer goat does were allocated into two groups comprising of 10 animals per group. For the Day 0 protocol, does oestrous cycles were synchronized for 9days and superovulated with pFSH starting 84h after the termination of progesterone treatment. For the traditional pFSH protocol, the oestrous cycle of does was synchronized for 9days, followed by superovulation with pFSH initiated 48h before CIDR withdrawal. For both groups does had two timed cervical inseminations with fresh undiluted semen. Embryos from both groups were flushed on day 6 following AI. The response to superovulation did not differ significantly between treatments but a tendency (P=0.06) was found for both fertilization and number of unfertilized ova in favour of the Day 0 protocol. The number of follicles 2–3mm, 4–5mm and total number of follicles at the beginning of a superovulation treatment was positively correlated to the total number of structures and embryos recovered. It is therefore concluded that the Day 0 protocol can be used for superovulation in Boer goat does however, more studies with large number of animals are recommended to ascertain its benefits. The correlation results suggest that the response to superovulation and quality of embryos recovered could be more determine by the size and number of follicles at the beginning of a superovulation treatment.

Effect of restricted suckling on the onset of follicular dynamics and body condition score in Brahman cattle raised under tropical conditions

With the aim of evaluating the effect of restricted suckling on the onset of follicular dynamics and body condition, multiparous Bos indicus cows were distributed in two groups. One group (RS=36) was subjected to a scheme of restricted suckling starting at 21days postpartum. Calves were allowed to suckle once per day for a period of two h whilst the control group (C=18) remained with their dams at all times. At calving, body condition score, back fat thickness and body weight had similar values (p>0.05) for both groups. By day 85 postpartum both groups had recorded losses in body weight. The cows in the continuous group formed a greater (p<0.05) number of follicles of class size <6mm in both periods before and after synchronization. The proportion of cows showing estrus and ovulation before 45days, was not different (p>0.05). The number of cows that exhibited estrus after 45days, was greater (p<0.05) in RS than C group, 72.2% and 55.5%, respectively. Same situation (p<0.05) occurred in cows that ovulated, 88.8 and 66.7%, for groups RS and C, respectively. The subset in the intensive observations showed that the size of the bigger follicle was larger (p<0.05) for RS cows than C cows from 36h after CIDR withdrawal. At 57h, the size of the biggest follicle recorded was not different between groups (p>0.05). A regime of restricted suckling favors the earlier growth of follicles and the prompt restoration of ovarian activity.

236 SUPEROVULATION IN SHEEP WITH REDUCED INJECTION FSH/eCG PROTOCOLS FOLLOWED BY TRANSFER OF FRESH OR VITRIFIED EMBRYOS

The aim of this experiment was to compare a single FSH/eCG, or double FSH-0.5% hyaluronan/eCG injection protocol with a multiple FSH/eCG injection protocol on the superovulatory response and embryo production in sheep. In addition, the effect of vitrification of these embryos on the pregnancy rate following embryo transfer was evaluated. Eighty Dohne Merino ewes received an 8-day CIDR-S device (0.33 g P4; Zoetis, Florham Park, NJ, USA) plus 10 mg of FSH i.m. (Folltropin-V; Vetoquinol, Lavaltrie, QC, Canada) and 400 IU of eCG i.m. (Pregnecol; Vetoquinol) in 3 treatment groups. Group 1 (n = 21) received a single 10-mg FSH injection in saline and 400 IU of eCG in saline 2.5 days before CIDR withdrawal. Group 2 (n = 23) received 6.7 mg of FSH in hyaluronan (MAP-5, 50 mg; Vetoquinol) and 400 IU of eCG in saline 2.5 days before CIDR withdrawal and 3.3 mg of FSH in hyaluronan 0.5 days before CIDR withdrawal. Group 3 (n = 36) received 7 injections (am, pm) of FSH in saline (2.5, 2.0, 1.5, 1.5, 1.0, 1.0, 0.5 mg) starting 2.5 days before CIDR withdrawal and 400 IU of eCG in saline at the first injection. Ewes were inseminated with semen collected from 1 of 5 rams at 36 to 40 h after CIDR withdrawal. Donor ewes were slaughtered 6 days after AI (Day 0), CL were counted and ova/embryos were collected. Viable embryos were transferred in singles into Day 6 synchronised recipients as either fresh (n = 128) or following vitrification/thawing using the CVM (CryoLogic, Blackburn, VIC, Australia; n = 97; Fry et al. 2005 Reprod. Fertil. Dev. 17, 243). Pregnancy was diagnosed by ultrasound scanning on Day 45. Data for CL and transferable embryo were analysed by the Kruskall-Wallis test and differences between groups determined by the Dunn test. Data for pregnancy rates was compared by chi-squared analysis. The mean number of CL in Groups 2 and 3 were both significantly higher than that in Group 1 (12.3 and 12.0 v. 8.5; P &lt; 0.05). Similarly, the total number of embryos/ova recovered in Groups 2 and 3 were significantly higher than for Group 1 (8.3 and 7.0 v. 5.0; P &lt; 0.05). Group 3 produced more viable embryos than either Group 2 or Group 1 (4.6 v. 2.7 and 2.1; P &lt; 0.05); however, data were skewed by the extensive use of semen from one ram in Group 2 that had a low fertilization rate (28%). The transfer of vitrified/thawed embryos resulted a nonsignificant (P &gt; 0.05) 10% decrease in pregnancy rate compared with fresh embryos (66% v. 76%). In conclusion, the administration of the sustained release FSH-MAP-5 in a 2-injection protocol in sheep was as effective as a multiple FSH injection protocol in inducing an ovarian response but more research is required to elucidate the effect of FSH-MAP-5 on embryo quality. The successful vitrification of sheep embryos provides a promising technique for the storage and transport of embryos in large-scale sheep embryo transfer programs.

13 EFFECT OF 6 TIMES REUSING OF CONTROLLED INTERNAL DRUG RELEASE (CIDR) FOR SHORT TERM (6 DAYS) ON PROGESTERONE LEVEL AND REPRODUCTIVE PERFORMANCE OF AWASSI EWES

This study was carried out using 120 multiparous Awassi ewes during breeding season to compare the effect of 6 times reusing of CIDR short term (6 days) on hormonal and reproductive performance. Ewes were equally and randomly allotted into 6 consecutive oestrus synchronization treatments. For group CIDR6×1, the ewes (n = 20) received new CIDR for 6 days with 300 IU of eCG at withdrawal time. For the other 5 groups, the same protocol was used, differing only by the use of the same CIDR for a second time in CIDR6×2, for a third time in CIDR6×3, for a fourth time in CIDR6×4, for a fifth time in CIDR6×5, and for a sixth time in CIDR6×6. Oestrus was detected using a vasectomized ram starting 12 h after progestagen withdrawal and repeated every 12 h up to 84 h. Blood samples were collected at withdrawal time (Day 6). Progesterone serum concentrations were measured using commercial ELISA kits and micro-titrimetric plates. Timed insemination was performed 48 h after CIDR withdrawal. Pregnancy and number of fetuses were diagnosed by ultrasonography at Day 23 after insemination and confirmed at Day 35 and 60. Comparisons among groups was evaluated using chi-square (χ2) test in all parameters except hormones levels, which was analysed by repeated-measures ANOVA, using SAS (SAS Institute Inc., Cary, NC, USA, 2000). The results revealed that there is no significant difference between the percentages of ewes detected in heat in groups CIDR6×1, CIDR6×2, and CIDR6×3 (55, 70, and 55%, respectively). While, the percentages of ewes detected in heat in group CIDR6×2 was significantly (P ≤ 0.05) higher than in groups CIDR6×4, CIDR6×5, and CIDR6×6 (35, 35, 31.58%, respectively). On the other hand, pregnancy rate was significantly (P ≤ 0.05) higher in CIDR6×2 (65%; 13/20) than CIDR6×3, CIDR6×4, CIDR6×5, and CIDR6×6 (30, 30, 30, and 15.79, respectively). No significant difference was detected in pregnancy rate between CIDR6×1 and CIDR6×2. No significant differences were detected in progesterone between CIDR6×1 and CIDR6×2, CIDR6×3, CIDR6×4, CIDR6×5, and CIDR6×6 (1.73, 1.67, 1.87, 1.57, 1.60, and 1.36 ng mL–1, respectively). These results indicated that reusing of CIDR devices for short term are efficient in synchronizing oestrus in ewes.

Effect of intermittent administration of neurokinin 3 receptor agonist on luteinizing hormone secretion, estrus, and ovulation in feed-restricted goats

The aim of this study was to determine the short-term effects of feed restriction on follicular dynamics and hormone secretion patterns in goats, and to examine the effect of senktide, a neurokinin 3 receptor agonist, under the feed-restricted condition. Goats were administered 2mg of dinoprost in the luteal phase of the estrous cycle, and an intravaginal progesterone device (CIDR) was inserted for 10 days. Feed was provided at the level of 50% (feed-restricted, n=12) or 100% maintenance (control, n=5) for 15 days, beginning on the day of CIDR insertion. At 12h after CIDR withdrawal, feed-restricted goats were intravenously administered with 200 nmol of senktide (n=6) or saline (n=6) at 4-h intervals for 24h. Fifteen days of feed restriction caused 12.4±5.5% body weight loss. On the day of CIDR withdrawal, feed-restricted goats had fewer follicles with smaller diameter than control goats. Feed restriction caused anestrus and anovulation in one case, and all the other goats in the control and feed-restricted saline-treated groups showed estrus and ovulated within normal ranges. Therefore, there was no significant influence of feed restriction on the timing of these events and related hormone profiles. In senktide-treated goats, there was a rapid increase in luteinizing hormone (LH) after each injection, which was followed by an increase in plasma estradiol concentration. In the senktide-treated group, 5/6 goats did not show estrus, and 3/6 goats ovulated 2 days after treatment, while most goats ovulated 3 or 4 days after treatment. These results suggest that feed restriction negatively affects follicular development and that senktide can enhance pulsatile LH secretion and thereby accelerate the ovulatory process.

Effects of calcium salts of fatty acids (CSFA) with different profiles (ω3 and ω6) during the flushing period on reproductive performance of ‘Afshari’ ewes

The effects of supplementing the flushing diet of Afshari ewes with calcium salts of fatty acids (CSFA) with different profiles (ω3 and ω6) on their reproductive performance were studied using 48 sheep weighing 46±3kg and 2–3 years old. The ewes were assigned to four groups (n=12 per group) and within a group received one of four diets for the period from the start of estrous synchronization to the end of mating (5 weeks). The diets all contained wheat bran (10%), wheat straw (37–40%), alfalfa (25–41%) and barley grain (7–25%). The control group received the basal (non-flushing) diet only. The remaining three groups received isoenergetic and isonitrogenous diets with higher energy contents formulated to meet the ewes’ requirements during the flushing period. In the Barley group, the basal diet was enhanced using additional barley grain; in the CaFl group the flushing diet included 5% of DM as flaxseed oil (ω3 CSFA); and in CaSf group the diet was supplemented with 5% of DM as sunflower oil (ω6 CSFA). The estrous cycles of the ewes were synchronized by application of a CIDR for 14 d and administration of 400 units of PMSG at CIDR withdrawal. Afshari rams were introduced to the ewes 36h later and the experimental diets continued to be fed for a further 3 weeks. The fertility and lambing rates were higher for Barley (91.7%; 125%), CaFl (100%; 150%) and CaSf (100%; 116.7%) than for the Control (83.3%; 83.3%). The number of lambs produced was significantly on the flushing treatments than the control. Serum cholesterol and progesterone levels were significantly higher on the diet supplemented with flaxseed oil (CaFl) than the other treatments. The results showed that supplementing the flushing diet with CSFA increased blood metabolites and hormones related to reproductive performance; and improved fertility, lambing rate and lamb birth weight. Ewes on the flaxseed oil supplemented flushing diet had the highest values for these variates. It was concluded that supplementing the flushing diet of ewes with flaxseed oil to provide a source of ω3 CSFA produces beneficial responses in their reproductive performance.

235 DEVELOPMENT OF IN VIVO-MATURED OOCYTES COLLECTED FROM JAPANESE BLACK CATTLE STIMULATED WITH DIFFERENT DURATIONS OF FOLLICULAR GROWTH

We demonstrated that in vivo-matured oocytes (mOC) collected by ovum-pick up (OPU) from cows after stimulation of follicular growth (FG) are suitable for producing good quality blastocysts (BL). However, it is not known whether duration of FG affects developmental competence of mOC. The purpose of this study was to examine development of mOC after stimulation with different duration of FG. Japanese black donor cows (n = 4 per each group), were treated with a CIDR at Day 0. Follicle of diameter &gt;8 mm were removed on Day 5. A total 20 AU of FSH was administrated to cows twice daily with decreasing doses from the evening of Day 6 to the morning of Day 10. In the conventional group (48PG), a administration of PGF2α (0.75 mg of cloprostenol), CIDR withdrawal, and administration of GnRH (0.2 mg of fertirelin acetate) were performed on the evening of Day 8, morning of Day 9, and morning of Day 10, respectively. In the experimental group (72PG), administration of PGF2a, CIDR withdrawal, and administration of GnRH were performed on the evening of Day 9, the morning of Day 10, and the morning of Day 11, respectively. The mOC were collected from follicles &gt;5 mm by OPU at 25 to 26 h following GnRH administration. Collected mOC were inseminated with 3 × 106 sperm mL–1 in BO solution on 30 h after GnRH. After 6 h of IVF, presumptive zygotes were cultured for 168 h in 5% CS + CR1aa, using a micro-well culture dish (Dai-Nippon-Print) and time-lapse cinematography (CCM-1.4MZS; Astec) for individual embryo observation. The kinetics of early embryo was analysis by CCM-1.4 software. To assess the quality of BL, prognostic factors were used as follows: (1) less than 27 hpi (hours post-insemination) at the first cleavage (1st CD), (2) 2 blastomeres at the end of 1st CD, and (3) absence of multiple fragments at the end of the 1st CD (Sugimura et al. 2012 PLoS ONE 7, e36627; Imai et al. 2014 Reprod. Fertil. Dev. 26, 182). Data were analysed by Student's t-test or chi-square test. The number of mOC were 12.5 ± 4.7 and 10.3 ± 2.7 (means ± s.e.) oocytes per session in 48PG and 72PG. There was no significant difference in cleavage rate or BL formation rate (97.5 ± 1.5 v. 98.2 ± 1.8%, 66.3 ± 8.2 v. 66.8 ± 3.5%, respectively). The time for 1st CD was shorter in 48PG (26.1 ± 0.3 v. 27.8 ± 0.4; P &lt; 0.01), and the rate of 1st CD less than 27 hpi was superior in 48PG compared with 72PG (74.3 v. 42.9%; P &lt; 0.05). However, the rate of 2 blastomeres and absence of multiple fragments were not different between 48PG and 72PG. The number of BL tended to decrease in 72PG compared with 48PG (28.6 v. 48.6%; P = 0.087). These results indicate that duration of FG did not affect the rate of cleavage and BL formation. However, extension of duration of FG might reduce the quality of BL.

The relationship between progesterone and biochemical constituents of amniotic fluid with placenta traits in Iranian crossbred ewes (Arkhar-Merino×Ghezel)

ObjectiveTo determine the values of amniotic fluid (AF) progesterone and biochemical constituents and its associated placenta traits in Iranian crossbred ewes (Arkhar-Merino×Ghezel). MethodsSixty ewes (2-5 years old, weighing 40-50 kg) were treated with controlled internal drug release induced 30 mg progesterone for 14 d and were injected with 400 IU pregnant mare serum gonadotropin at the time of CIDR withdrawal. After the detection of estrus, ewes were hand-mated. After expelling of each fetus accompanied by fetal membranes, 10 mL AF was taken from fetal sac for spectrophotometer methods (glucose, urea, creatinine, total protein, cholesterol, triglycerides, calcium and phosphorus) and radioimmunoassay (progesterone) analysis. ResultsResults indicated that there were highly positive correlations between placental efficiency and cotyledon density (r=0.764, P<0.01), and negative correlation between placental weight (PW) with the total volume amniotic fluid (TVAF) (r=−0.872, P<0.01). A positive relationship was calculated between TVAF with total protein and creatinine (r=0.418, P<0.01 and r=0.639, P<0.05, respectively). However, the correlation between the glucose and PW, cotyledon length and calcium were significantly positive (r=0.704 and r=0.712; P<0.01, respectively). The findings of this study demonstrated that there were no relationships between progesterone concentrations and placental traits (P>0.01), except for PW (r=0.665). ConclusionsIn conclusion, the significant correlation between the AF biochemical and progesterone with placental traits for the above-mentioned metabolites suggests that metabolic changes in AF levels will reflect the condition in the AF.

The requirement of GnRH at the beginning of the five-day CO-Synch + controlled internal drug release protocol in beef heifers.

The objective of this study was to determine if the omission of GnRH at controlled internal drug release device (CIDR) insertion would impact pregnancy rates to timed AI (TAI) in beef heifers enrolled in a 5-d CO-Synch + CIDR protocol that used 1 PGF2α dose given at CIDR removal. Yearling beef heifers in Ohio in 2 consecutive breeding seasons (2011, n = 151, and 2012, n = 143; Angus × Simmental), Utah (2012, n = 265; Angus × Hereford), Idaho (2012, n = 127; Charolais), and Wyoming (2012, n = 137; Angus) were enrolled in the 5-d CO-Synch + CIDR protocol. At CIDR insertion (d -5), heifers were randomly assigned either to receive 100 μg GnRH (GnRH+; n = 408) or not to receive GnRH (GnRH-; n = 415). At CIDR removal (d 0 of the experiment), 25 mg PGF2α was administered to all heifers. All heifers were inseminated by TAI and given 100 μg GnRH 72 h after PGF2α (d 3). In heifers at the Ohio locations (n = 294), presence of a corpus luteum (CL) at CIDR insertion (d -5) was determined via assessment of progesterone concentrations (2011) and ovarian ultrasonography (2012). Subsequently, in both years, ovarian ultrasound was conducted on d 0 to determine the presence of a new CL. In this same subgroup of heifers, blood samples for progesterone analysis were collected on d 3 to assess luteal regression. Pregnancy diagnosis was performed between 32 and 38 d after TAI. At CIDR withdrawal, presence of a new CL was greater (P < 0.05) in the GnRH+ (55.8%, 82/147) than GnRH- (26.5%, 39/147) treatment. Incidence of failed luteal regression did not differ between the GnRH+ (3.4%) and GnRH- (0.7%) treatments. Pregnancy rate to TAI did not differ between the GnRH+ (50.5%) and GnRH- (54.9%) treatments. In conclusion, although the incidence of a new CL at CIDR removal was increased in the GnRH+ treatment, omission of the initial GnRH treatment in the 5-d CO-Synch + CIDR protocol did not influence TAI pregnancy rate in yearling beef heifers. In addition, a single dose of PGF2α at CIDR removal was effective at inducing luteolysis in yearling beef heifers enrolled in the 5-d CO-Synch + CIDR protocol, regardless of whether or not the initial GnRH treatment was given.

Effect of interval from induction of puberty to initiation of a timed AI protocol on pregnancy rate in Nellore heifers

Prepubertal Bos indicus heifers (n = 774) were submitted to an E2/P4-based timed artificial insemination (TAI) protocol at three different intervals after induction of their pubertal ovulation by insertion of an intravaginal progesterone (P4) device for 12 days. Heifers were randomly assigned to start the TAI protocol at 10 (group 10; n = 253), 12 (group 12; n = 265), or 14 (group 14; n = 256) days after the P4 device was removed. The TAI protocol consisted of the following: insertion of intravaginal device containing P4 (Controlled internal drug release [CIDR]; previously used twice for 9 days each) + estradiol benzoate (2 mg) on Day 0, CIDR withdrawal + estradiol cypionate (0.5 mg) and PGF2α (12.5 mg) on Day 9, and TAI on Day 11. A subgroup of heifers (n = 472) was evaluated by ultrasound on Days 9 and 11 to evaluate the ovaries and to determine P4 concentrations on Day 9. On Day 9, more (P < 0.05) CLs were present, and follicular diameter was smaller (P < 0.05) for group 10 than for groups 12 and 14 (38.4%, 29.3%, and 23.3% with CL and 9.4 ± 0.1, 9.9 ± 0.1, and 9.8 ± 0.1 mm diameter, respectively), but P4 concentrations did not differ (P > 0.1) between treatments (2.4 ± 0.06 ng/mL). Follicular diameter at TAI (11.08 ± 0.09 mm) and ovulation rate (88.4%) did not differ between treatments (P > 0.1). However, conception and pregnancy rates for all heifers were greater (P < 0.05) in group 12 (50.4% and 45.5%, respectively) than in group 10 (38.2% and 33.7%, respectively), with group 14 intermediate to other treatments (45.6% and 40.6%, respectively). The final pregnancy rate did not differ between treatments (80.9%). In conclusion, a 12-day interval from the end of the puberty induction protocol to the start of the TAI protocol resulted in greater conception and pregnancy rates in prepubertal Nellore heifers.

Long-acting neuroleptic use for reproductive management of non-domestic ungulates using the domestic goat (Capra hircus) as a model

Fluphenazine decanoate is a long-acting phenothiazine neuroleptic that attenuates the stress response and may be useful during intensive handling for reproductive procedures in non-domestic ungulates. However, phenothiazines are also associated with elevated serum prolactin, which can suppress fertility in some species. For this study, 10 female domestic goats were used as a model for non-domestic caprids to test effects of fluphenazine decanoate on serum cortisol and reproductive cyclicity following estrus synchronization. Two identical trials were conducted during the breeding season, employing a random crossover design. First, females underwent estrus synchronization using a 14-day treatment with progesterone (330 mg; CIDR). After 7 days of CIDR exposure, the treatment group (n = 5) received fluphenazine decanoate (1.0 mg/kg IM) and controls (n = 5) received an equivalent volume of 0.9% saline IM. At CIDR withdrawal (Day 14), animals received 125 mg cloprostenol sodium to lyse any luteal tissue and synchronize estrus. Blood was collected every 2 hr from 36 hr after CIDR withdrawal until 24 hr after standing estrus, or up to 5 days to monitor stress and reproductive hormones. Serum cortisol, prolactin, luteinizing hormone (LH) and progesterone concentrations were determined by enzyme immunoassay. While treatment with fluphenazine was associated with lower cortisol concentrations compared to controls (P = 0.001), 4 of the 10 treated animals experienced elevated serum prolactin, suppression of the LH surge and inhibition of ovulation. These findings suggest that long-acting neuroleptic drugs reduce the adrenal stress response, but may interfere with reproductive responses and negatively influence breeding success.

213 USE OF ACTIVE CASPASE 3 AND TUNEL ASSAYS TO ESTIMATE EMBRYONIC QUALITY IN IN VIVO SANTA INES EWE EMBRYOS

This study was designed to quantify the percentage of apoptotic cells using active caspase 3 and TUNEL assays, in order to estimate the quality of ovine embryos produced in vivo. For that, 60 Santa Ines ewes (n = 10 per group) were submitted to superovulation with FSH treatment started near the different follicular wave emergence of the protocols (G-1 or G-2), during breeding season, transition, and nonbreeding season. Follicular wave emergence days were defined in a previous study that evaluated the follicular dynamic in oestrus synchronization treatments (Oliveira et al. 2011 Acta Sci. Vet. 38, 361). On Day 0, all ewes received a P4 device (CIDR®) and 37.5 μg of D-cloprostenol. The P4 device was replaced by a new one on Day 7 just for G-2 in the transition period. The superovulation treatment started on Day 4, 4, and 6 of protocol for G-1 and on Day 10, 10, and 11 for G-2 in nonbreeding, transition, and breeding season, respectively. The FSH treatment consisted of eight injections of pFSH administrated twice a day in descending order (40, 30, 20, and 10 mg of pFSH). The P4 device was removed two days after beginning of FSH treatment. At these times, all ewes received another injection of 37.5 μg of D-cloprostenol and a dose of 200 IU of eCG. Ewes were mated with a fertile ram for 4 days after P4 device removal. Embryo recoveries were carried out by laparotomy, 7 days after CIDR withdrawal. Embryos were morphologically classified. Grade I to III morulas and blastocysts were considered viable. A representative sample of each treatment was fixed and stained by active caspase 3 and TUNEL assays to assess the apoptotic cells percentage. Data were analysed by GLIMMIX using SAS comparing mean values (±s.e.m.) between groups at each season (P = 0.05). Pearson correlation was estimated between active caspase 3 and TUNEL assays. No effect was detected between treatments in each season on the number of viable embryos (3.2 ± 0.8 v. 1.8 ± 0.8, 3.9 ± 1.9 v. 5.7 ± 1.4, and 3.8 ± 1.5 v. 3.4 ± 0.8 for G-1 v. G-2 in nonbreeding, transition and breeding season, respectively). The treatment G-2 increased (P &lt; 0.05) apoptotic cells percentage in nonbreeding season group, assessed by active caspase 3 (G-1: 3.1 ± 1.6% and G-2: 12.8 ± 4.3%) and TUNEL (G-1: 1.6 ± 0.5% and G-2: 11.1 ± 3.5%) assays. The apoptotic cells percentage remained unaltered for Transition and Breeding season groups, assessed by either active caspase 3 (G-1: 6.0 ± 0.9% and 5.6 ± 1.5%; G-2: 5.6 ± 1.1% and 5.1 ± 0.5%) and by TUNEL (G-1: 7.5 ± 1.3% and 5.2 ± 1.0%; G-2: 5.0 ± 0.9% and 6.4 ± 1.1%). The Pearson correlation between active caspase 3 and TUNEL tests was r2 = 0.436 (P &lt; 0.0001). In conclusion, the active caspase 3 and TUNEL assays presented similar results for apoptosis level assessment in Santa Ines ewes in vivo produced embryos, and both assays were considered appropriate for this purpose. The increased apoptosis levels detected in the G-2 nonbreeding season group suggest that this treatment is harmful for Santa Ines ewe embryos. Financial support was provided by FAPESP and FUNDUNESP.

Response to the buck effect in goats primed with CIDRs, previously used CIDRs, or previously used autoclaved CIDRs during the non-breeding season

The sudden introduction of males induces ovulation in anestrous goats. However, unless goats are primed with progestagens, in most cases ovulation is followed by short cycle, decreasing the conception rate. CIDR devices may be used for more than one treatment, but as progesterone content decrease, estrous and conception rates are negatively affected. It has been reported in cattle that progesterone concentrations increase after autoclaving the devices. The aim of the present study was to determine the plasma progesterone levels, and the estrous and pregnancy rates of anestrous goats previously primed with a new CIDR, a previously used CIDR or a previously used and autoclaved CIDR and exposed to the male effect. During April–May, a total of 126 adult dairy goats were treated for 5 days with a new CIDR (NC, n=40), a previously used and disinfected CIDR (DUC, n=44), and a previously used and autoclaved CIDR (UAC, n=42). Recycled devices were previously used for 14 days. At CIDR withdrawal, does were joined to sexually experienced adult marking males. Goats in estrus were recorded during 10 days, and pregnancy was determined by ultrasound 50 days after CIDR withdrawal. Blood progesterone concentrations were measured throughout the treatment. Goats treated with a new CIDR had greater progesterone concentrations than DUC (P<0.05), but similar than UAC goats (P>0.05). Progesterone concentrations tended to be significantly greater in group UAC than in group DUC (P=0.06). The area under the curve of progesterone was greater in groups NC (25.4±2.6ngdaymL−1) and UAC (18.1±2.6ngdaymL−1) than in DUC (11.0±2.6ngdaymL−1, P<0.05). Estrous (97.5, 100 and 100%) and pregnancy rates (62.5, 79.5 and 69%) were not different between groups (NC, DUC and UAC respectively). In conclusion, autoclaving used CIDRs, after a previous use of 14 days, tended to increase progesterone concentrations in treated goats. Estrus and fertility in goats exposed to the buck effect was not affected by the use of primings with new CIDRs, previously used CIDRs or previously used autoclaved CIDRs.

Fertility response with four estrus synchronization regimens prepubertal buffalo heifers

The use of four estrus synchronization regimens in 43 prepubertal buffalo heifers revealed that progesterone-based hormone regimens (ovsynch+CIDR±eCG) are most favourable for the optimization of their fertility status. Treatment with eCG at CIDR withdrawal had no additional beneficial impact on conception rate. Under field conditions, it may difficult to predict exhibition of induced-estrus/ ovulation in prepubertal heifers, therefore, heifers subjected to Ovsynch+CIDR regimen should be inseminated at 48 (day 9), 72 (day 10) and 96 (day 11) h after CIDR withdrawal.